Polyvalent conjugate vaccine for cancer

ABSTRACT

This invention provides a polyvalent vaccine comprising at least two conjugated antigens selected from a group containing glycolipid antigen, polysaccharide antigen, mucin antigen, glycosylated mucin antigen and an appropriate adjuvant. This invention also provides a multivalent vaccine comprising at least two of the following: glycosylated MUC-1-32mer, Globo H, GM2, Le y , Tn(c), sTN(c), and TF(c). This invention provides the vaccine above, wherein the adjuvant is saponin-based adjuvant. This invention provides a method for inducing immune response in a subject comprising administering an effective amount of the vaccine above to the subject. Finally, this invention provides a method for treating cancer in a subject comprising administering an appropriate amount of the vaccine above to the subject.

The application disclosed herein is a continuation-in-part ofInternational Application No. PCT/US02/21348, filed Jul. 5, 2002, whichclaims priority of U.S. Ser. 60/303,494, filed on Jul. 6, 2001 and U.S.Ser. No. 60/347,231, filed on Jan. 10, 2002, the contents of which arehereby incorporated by reference into this application.

The invention disclosed herein was made with United States governmentsupport under NIH Grant Nos. CA33049 and CA52477 from the United StatesDepartment of Health and Human Services. Accordingly, the United StatesGovernment has certain rights in this invention.

Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

Tumor-specific antigens have been identified and pursued as targets forvaccines. Previous work from the inventors' has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safewith local erythema and edema but minimal systemic toxicities. As aresult of vaccination with these monovalent vaccines, most patientsgenerated specific high titer IgM or IgG antibodies against therespective antigen-KLH conjugates. The present invention provides apolyvalent vaccine wherein the components of the monovalent vaccines arecombined and administered with an adjuvant as treatment for cancer.

SUMMARY OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprisingat least two conjugated antigens selected from a group containingglycolipid antigen, polysaccharide antigen, mucin antigen, glycosylatedmucin antigen and an appropriate adjuvant. This invention also providesthe multivalent vaccine, comprising glycosylated MUC-1-32mer, Globo H,GM2, Le^(y), Tn(c), and TF(c). This vaccine may comprise glycosylatedMUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c). This inventionprovides the vaccine above, wherein the adjuvant is saponin-basedadjuvant.

This invention also provides a method for inducing immune response in asubject comprising administering an effective amount of the vaccineabove to the subject. Finally, this invention provides a method fortreating cancer in a subject comprising administering an appropriateamount of the vaccine above to the subject.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides a polyvalent vaccine comprisingat least two conjugated antigens selected from a group containingglycolipid antigen, polysaccharide antigen, mucin antigen, glycosylatedmucin antigen and an appropriate adjuvant.

The glycolipid includes but is not limited to Globo H, a Lewis antigenand a ganglioside. The Lewis antigen includes but is not limited toLe^(y) and sialyl Le^(a). The ganglioside includes fucosylated GM1, GM2,GD2, or GD3. In another embodiment, the mucin is a MUC peptide. In afurther embodiment, the MUC peptide is MUC-1, MUC-2 or MUC-16. Thepolysaccharide antigen includes but is not limited to Tn(c), sTn(c),TF(c), and polysialic acid.

This invention provides a bivalent, trivalent, tetravalent, pentavalent,hexavalent, and heptavalent vaccine. The vaccine comprises at least twoconjugated antigens selected from a group containing glycolipid antigen,polysaccharide antigen, mucin antigen, glycosylated mucin antigen and anappropriate adjuvant.

In an embodiment, the hexavalent vaccine comprises glycosylatedMUC-1-32mer, Globo H, GM2, Le^(y), Tn(c), and TF(c). In a furtherembodiment, the range of MUC-1-32mer is from about 0.1 to 30 ug. In yetanother embodiment, the range of Globo H is from about 0.1 to 100 ug. Instill a further embodiment, the range of GM2 is from about 0.1 to 100ug. In an additional embodiment, the range of Le^(y) is from about 0.1to 60 ug. In a further embodiment, the range of Tn(c) is from about 0.1to 100 ug. In an additional embodiment, the range of TF(c) is from about0.1 to 30 ug.

In a separate embodiment, the adjuvant is saponin based. The adjuvantincludes QS21 and GPI-0100. In an embodiment, the range of QS21 is fromabout 25 to about 200 ug. In another embodiment, QS21 is about 100 ug.In a separate embodiment, the adjuvant is GPI-0100 with a range fromabout 1 to 25 mg. In an embodiment, GPI-0100 is about 10 mg.

This invention provides a heptavalent vaccine comprising at least twoconjugated antigens selected from a group containing glycolipid antigen,polysaccharide antigen, mucin antigen, and glycosylated mucin antigenand an appropriate adjuvant. In an embodiment, the vaccine comprisesglycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTN(c), and TF(c).In another embodiment, the range of MUC-1-G5 is from about 0.1 to 30 ug.In a further embodiment, the range of Globo H is from about 0.1 to 100ug. In another embodiment, the range of GM2 is from about 0.1 to 100 ug.In still another embodiment, the range of Le^(y) is from about 0.1 to 60ug. In an embodiment, the range of Tn(c) is from about 0.1 to 100 ug. Ina further embodiment, the range of sTn(c) is from about 0.1 to 100 ug.In yet another embodiment, the range of TF(c) is from about 0.1 to 30ug.

This invention provides the vaccine above, wherein the adjuvant issaponin-based adjuvant. These saponin-based adjuvants include but arenot limited to QS21 and GPI-0100.

In an embodiment, the range of QS21 is from about 25 to 200 ug. Inanother embodiment, the QS21 is about 100 ug. In a separate embodiment,the adjuvant is GPI-0100 and the range is from about 1 to 25 mg. In apreferred embodiment, GPI-0100 is about 10 mg.

This invention provides a polyvalent vaccine comprising a conjugatedglycosylated antigen, a conjugated ganglioside antigen and anappropriate adjuvant, wherein the antigens are conjugated to a carrier.In an embodiment, the carrier is Keyhole Limpet Hemocyanin (KLH).

This invention provides the polyvalent vaccine above comprising at leasttwo conjugated antigens selected from a group containing glycolipidantigen, polysaccharide antigen, mucin antigen, and glycosylated mucinantigen and an appropriate adjuvant for cancer. In an embodiment, thecancer is prostate, breast or ovarian cancer.

This invention also provides a method for inducing immune response in asubject comprising administering an effective amount of the abovevaccine to the subject.

Furthermore, this invention provides a method for treating cancer in asubject comprising administering an appropriate amount of the abovevaccine to the subject.

This invention also provides a composition comprising the above vaccineand a carrier.

This invention also provides a pharmaceutical composition comprising theabove vaccine and a pharmaceutically acceptable carrier.

In addition, the invention provides a vaccine for small cell lung cancercomprising at least two conjugated antigens selected from the groupcontaining Globo H, fucosylated GM1, GM2, GD2, GD3, sialyl Le^(a) andpolysialic acid. This invention also provides a method for inducingimmune response in a subject bearing small cell lung cancer comprisingadministering an effective amount of the above vaccine to the subject.This invention furthermore provides a method for treating a subjectbearing small cell lung cancer comprising administering an effectiveamount of the above vaccine to the subject.

In addition, this invention provides the above vaccine, furthercomprising an antigen selected from a group containing CA125, or aportion thereof, KSA peptide or protein, and PSMA, or a portion thereof.

This invention includes the above vaccines which further comprise otherantigens which can induce antibody and/or immune response. Asillustrated throughout the specification, the antigen used may bemodified to increase its immunogenicity. Said antigens include but arenot limited to CA125, or a portion thereof, KSA peptide or protein, andPSMA, or a portion thereof. As can be easily appreciated by the ordinaryskilled artisan, only a portion of the antigen may be required forinduction of immune response from a subject.

As stated herein, subjects are organisms which have immune response. Thesubject includes but is not limited to humans. Said subject could bedomestic animals, such as dogs and cats.

This invention further provides the above compositions and apharmaceutically acceptable carrier, thereby forming pharmaceuticalcompositions.

This invention also provides a pharmaceutical composition comprising acombination as described above and a pharmaceutically acceptablecarrier. For the purposes of this invention, “pharmaceuticallyacceptable carriers” means any of the standard pharmaceutical carriers.Examples of suitable carriers are well known in the art and may include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution and various wetting agents.Other carriers may include additives used in tablets, granules andcapsules, etc. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gum, glycols or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well-known conventionalmethods.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

Experimental Details First Series of Experiments Polyvalent (Hexavalent)Conjugate Vaccine for Prostate, Breast, Ovarian and Small Cell LungCancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), or TF(c) conjugated to KLH to be safe withlocal erythema and edema but minimal systemic toxicities. As a result ofvaccination with these monovalent vaccines, most patients generatedspecific high titer IgM or IgG antibodies against the respectiveantigen-KLH conjugates. The present invention provides a hexavalentvaccine wherein the components of the monovalent vaccines are combinedand administered with an adjuvant as treatment for prostate, breast,ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of six tumor antigensadministered with a saponin immunological adjuvant QS-21 or GPI-0100.The antigens are glycosylated MUC-1-32mer, Globo H, GM2, Le^(y), Tn(c),and TF(c). In each case the antigen is conjugated to Keyhole LimpetHemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-32mer: 0.1 to 30 μg;    -   Globo H: 0.1 to 100 μg;    -   GM2: 0.1 to 100 μg;    -   Le^(y): 0.1 to 60 μg;    -   Tn(c): 0.1 to 10 μg;    -   TF(c): 0.1 to 30 μg;    -   QS-21: 100 μg;    -   GPI-0100: 1 or 25 mg.

Example 1 A Phase I Multivalent Conjugate Vaccine Trial for Patientswith Biochemically Relapsed Prostate Cancer 1.0 PROTOCOL SUMMARY 2.0OBJECTIVE 3.0 BACKGROUND AND RATIONALE 4.0 VACCINE PREPARATION 5.0IMMUNIZATION SCHEDULE 6.0 PRE- AND POST-THERAPY EVALUATION 7.0 RESPONSECRITERIA 8.0 BIOSTATISTICAL CONSIDERATIONS 9.0 REFERENCES 1.0 PROTOCOLSUMMARY

This is a phase I pilot trial designed to assess safety using amultivalent conjugate vaccine. This trial is based on the results ofeight dose-seeking phase I monovalent glycoprotein and carbohydrateconjugate vaccine trials which have been shown to be consistentlyimmunogenic in man. These trails also allowed us to screen candidateantigens for their ability to generate high titer specific antibodiesagainst the immunizing antigen. This vaccine will consist of the highestdose of synthetic glycoprotein and carbohydrate antigens shown to elicithigh titer IgM and IgG antibodies in patients with biochemicallyrelapsed prostate cancer. The inventors' previous work has shown themonovalent vaccines to be safe with local erythema and edema but minimalsystemic toxicities. Among the antigens to be included in themultivalent vaccine are carbohydrate antigens Globo H and GM2 and theglycoprotein antigens glycosylated MUC-1-32mer, Lewisy, Tn(c), andTF(c). The patient populations to be targeted are those patients whohave failed primary therapies such as prostatectomy or radiation or havebeen on intermittent hormonal therapy and have remained hormonallysensitive in the absence of radiographic disease. These populations musthave as the sole indication of disease progression, a rising PSA. Theinventors' data from approximately 160 men who participated in earliermonovalent vaccine trials against the aforementioned antigens have shownthat a treatment effect in the form of a decline in PSA log slopescompared with pretreatment values could be seen in patients with minimaltumor burden. A phase III double blind randomized trial with two hundredforty patients is planned based on the safety data accrued form thisproposed phase I trial. The primary endpoint of the study will be theability to assess the safety of the vaccine and the humoral response toa multivalent conjugate. Secondary endpoints will be to evaluatepost-therapy changes in PSA.

2.0 OBJECTIVES 2.1 The Primary Endpoints of the Study are:

2.1.1 To determine the safety of a multivalent conjugate vaccine inpatients with prostate cancer who have biochemically relapsed followingprimary therapies such as surgery or radiation.2.1.2 Measure the antibody response against the individual components ofthe vaccine and to correlate the response to subsequent clinical course.2.2. The secondary endpoints will be:2.2.1 To assess post-immunization changes in prostate specific antigenlevels and other objective parameters of disease (radionuclide bone scanand/or measurable disease if present.

3.0 BACKGROUND AND RATIONALE 3.1 Prostate Cancer:

Over 180,000 cases of prostate cancer will be diagnosed in the UnitedStates in 2000.¹ Of these, 30-35% will present with tumors beyond theconfines of the gland, while an additional 25% will develop metastasesin the course of the disease despite local therapies. In these cases, arising PSA antedates the development of overt metastases by a median of12-24 months. Androgen ablation is the standard treatment with upwardsof 70% of cases showing a normalization of an abnormal PSA aftertherapy. When to initiate treatment remains an area of controversy andthere is no evidence that deferring therapy compromises outcomes. Thisobservation, coupled with the fact that most patients relapsed within amedian of 12-18 months², and that most men can not tolerate the sideeffects of castration including impotency, weight gain and hot flashes,has led to the search for alternative therapies. One such approachinvolves enhancing the body's own immune system as a means to treatlocal disease and prevent disease progression. PSA monitoring allows theidentification of patients with low-volume disease, in whom animmunostimulatory approach may be more efficacious relative to a heavilypretreated, symptomatic population with large tumor burdens.Vaccinations represent a safe intervention with minimal toxicities thatcan be given as an adjuvant to surgery or radiation therapy in men atrisk for systemic relapse. They can also be offered to men with minimaltumor burdens who are progressing and who are not willing to accepttoxicities of hormonal therapy or chemotherapy. Because hormonal statusmay effect antigen expression and regulation, we propose to enrollpatients with different hormone sensitivities. This will includepatients who have not received hormonal therapy or have been onintermittent hormonal therapy.

3.2 PSA as an Endpoint for Clinical Trials:

The availability of serum PSA determinations provides a unique trialdesign for testing new therapies rapidly as changes in PSA levels overtime correlate well with clinical outcomes.³ This relationship holds forboth hormone-naïve and hormonally relapsed disease. Once sequentialelevations in PSA are documented in the setting of castrate testosteronelevels, clinical symptoms develop in a median of 3-6 months. Thisobservation justifies treatment in the setting of rising PSA values,using post-therapy changes in PSA as the outcome measure. With thisdesign, therapeutic approaches that do not produce a defined degree ofdecline in PSA on multiple determinations for a defined duration (videinfra) are not evaluated further.²

3.3 Immunologic Approaches:

Augmentation of the immune response to cancer can be attempted by twobasic approaches: non-specific immunopotentiation which constitutes thebulk of past and current efforts at cancer immunotherapy, and specificimmunization which has not really been evaluated in the treatment ofcancer but has contributed much to the control of infectious diseases.It is the knowledge of microbial antigens which has permitted thedevelopment of successful specific immunization against infections. Thelack of availability of well-defined human cancer antigens, on the otherhand, has prevented exploration of specific immunization in the contextof cancer as it should be explored, using vaccines of definedcancer-restricted antigenicity and demonstrating their immunogenicity incancer patients.

3.3.1 The Role of Carbohydrates and Mucins in Prostate Cancer:

Carbohydrate antigens have proven to be clinically relevant and (asidefrom vaccines against toxins) are the only defined bacterial antigensused in vaccines against bacterial pathogens. Immunization withcarbohydrate antigens has also resulted in directed antibody responsesagainst human tumor cells (reviewed in ⁴), presumably because theseantibodies are known to mediate antibody-dependent cell-mediated andcomplement mediated lysis of tumor cells, complement-inducedinflammation, and phagocytosis by the reticulo-endothelial system. Theinventors' previous study in prostate cancer focused on defining theantigens expressed on the surface of prostate cancer cells.3.3.2 Results with Eight Monovalent Phase I Trials Using GlycoproteinPeptides and Carbohydrate Antigens.Immunohistochemistry using well-defined monoclonal antibodies againstglycoprotein and carbohydrate antigens have shown that primary andmetastatic prostate carcinoma specimens express these heretofore unknownantigens and that these molecules can serve as targets for immunerecognition. We have studied two mucin peptide antigens, MUC-1 and MUC-2conjugated to KLH and given with the immune adjuvant, QS21 in the phaseI setting as a dose escalating trial with 10, 30 100 and 3 μg. Patientsreceived five subcutaneous vaccines over the course of twenty-six weeksat weeks 1,2, 3, 7, and 19. Twenty patients were treated in theMUC-1-KLH-QS21 trial and fifteen were treated with MUC-2-KLH-QS21 trial.All patients developed high titer IgM and IgG antibodies specific forthe immunizing peptide. Antibody titers rose by week 7 and declinedusually by week 19, the time of the fifth and final vaccine.Unexpectedly, a treatment effect was observed after the vaccine trialwas completed in the form of a declining PSA log slope compared withpretreatment values in approximately two-thirds of patients. In manypatients, the slope began to show a decline by week 38 with subsequentdeclines by week 60. The initial decline corresponded to the rise ofantibodies following the last immunization received at week 19. Fivepatients who were treated with the MUC-1-KLH conjugate in 1996 continueto have stable PSA log slopes without radiographic evidence of disease.Patients who were treated with MUC-2-KLH conjugate also demonstrated asimilar treatment effect, however, the trial has not as yet reachedmaturity. The vaccines were found to be safe with erythema, tendernessand edema at the injection site. No evidence of autoimmunity or systemictoxicity was observed.3.3.3 Experience with Glycolipid and Carbohydrate Antigens.Eighteen patients have undergone immunization with Globo H, a glycolipidantigen expressed on prostate cancer cells. This is the first purelysynthetic complex carbohydrate antigen used for immunization in mancapable of generating high titer specific antibodies (median peak titer1:320, IgG median titer 160) capable of mediating complement lysis oftumor cells. Several patients generated IgM antibody titers of1:20,480). Patients were immunized with 10, 30 100 or 3 μg of GloboH-KLH plus QS21 over twenty-six weeks. Of the patients immunized withthis vaccine, six remain active with stable PSA log slopes and noradiological evidence of evidence of disease over the last 2½ years.This vaccine was found to be safe with no evidence of systemic toxicity.Ganglioside antigens (acidic glycosphingolipids expressing sialic acidat one end and ceramide at the other) was also investigated in a trialcomparing the immunogenicity of higher doses of QS21. Using GM2-KLH at30 μg, a dose previously established in melanoma trials, 18 patientswere immunized with either the GM2 conjugate plus QS21 at the standarddose of 100 μg or QS21 at 225 μg. Because of its potential for systemictoxicity, the latter vaccine was given as three separate immunizationsto three separate sites as GM2-KLH at 10 μg plus QS21 at 75 μgsubcutaneously. No difference in antibody titers were observed in twogroups of patients; although two patients from the group given thehigher dose of QS21 experienced grade II myalgias. Several patients alsoexhibited a decline in PSA log slopes but there did not appear to be anydifference between groups with regard to treatment effects.

4.0 VACCINE PREPARATION

Globo H, Lewis^(y), Tn(c), TF(c) are synthesized in the laboratory ofBio-Organic Chemistry headed by Dr. Samuel Danishefsky. MUC-1-32mer issynthesized in the Core Peptide Synthesis Facility of The RockefellerResearch Laboratories under the aegis of Dr. Paul Tempst. It isglycosylated with Tn by Dr. Henrik Clausen at the University ofCopenhagen, Copenhagen, Denmark. GM2 is extracted from rabbit brains byProgenics, Inc., Tarrytown, N.Y.4.1 Globo H, MUC-1-32mer, GM2, Lewis^(y), Tn(c) and TF(c)-KLHConjugation:The above antigens will be covalently attached to KLH in Dr.Livingston's laboratory. Antigen-KLH ratios between 150/1 and 800/1assuming a KLH molecular weight of 5×10⁶ will be accepted. Gels will beperformed and western blot analysis will be conducted with each lot ofantigen-KLH for comparison to future lots. Sterility and safety testingwith each lot plus QS21, at >50 times the dose/meter² to be used inclinical trials will be performed. No growth in culture and no adversereaction in mice or Guinea pigs (including weight loss of 10% or more)will be tolerated. Two or more mice will be immunized with eachantigen-KLH batch on 2-3 occasions at 1-2 week intervals and postimmunization sera tested. Antibody titers of 1/200 or greater againstantigen and 1/40 by IA or FACS staining of >25% of antigen positivecells will be accepted as proof that the construct has the appropriateimmunogenicity.

4.2 Antigen Doses:

Based on previous vaccine trials in prostate cancer patients, thefollowing doses have been established for the multivalent trial:glycosylated MUC-1-32mer, 3 μg; Globo H, 10 μg; GM2, 10 μg; Le^(y), 10μg; Tn(c), 3 μg; and TF(c), 3 μg. QS21 will be used at 100 μg as nosignificant difference in immunogenicity was observed with doses as highas 225 μg.

4.3 Safety Testing:

Samples from the materials are sent for sterility and safety testing.Immunogenicity of the individual peptides/carbohydrates have beenpreviously confirmed in mice.

5.0 IMMUNIZATION SCHEDULE 5.1 Patient Selection:

All patients with evidence of biochemical relapse will be considered.Hormonal status will be recorded on the basis of serum testosteronelevels as follows: Patients who have progressed after primary surgery orradiation (with or without neo-adjuvant androgen ablation) who havenon-castrate levels of testosterone (>50 ng/ml) will be eligible.

5.2 Interval:

The immunization schedule that we will utilize was derived from theinventors' studies with other glycoprotein and carbohydrate conjugatevaccines in patients with melanoma, colon and breast cancers.

5.3 Treatment Schedule and Dose:

Fifteen patients will be treated with specified doses of eachcarbohydrate or peptide constituent as has been determined previouslybased on earlier monovalent trials completed. QS21 will be administeredat the standard dose of 100 ug. Sites: The vaccine conjugate will beadministered subcutaneously to random sites on the upper arms and upperlegs.

5.4 Dose Modifications:

If a patient experiences a Grade III or greater local or Grade II orgreater systemic toxicity at any time a decrease by 50% in allcomponents of future vaccinations will be administered for that patient.

6.0 PRE- AND POST-THERAPY EVALUATION 6.1 Outcomes:

The study evaluation will include parameters to assess the safety of thevaccine, antitumor effect, as well as assessments of immune function.Interval safety assessments will include the Patient Diary. An overallantitumor assessment will be performed during weeks 13 and 26. If thepatient has not demonstrated progression of disease at week 13 or 26, hewill continue on protocol. Upon completion of the trial, he will bemonitored every 3 months with bloodwork and imaging studies for the next2 years or until disease progression.

6.2 Safety and Antitumor Effects:

STUDY WEEK O^(a, c) 1 2 3 7 9 13 19 26 Clinical: Performance StatusX^(b) — — X X X X X X^(h) Interval Hx & PE X^(b) — — X X X X — X CBC,Diff, Plt. X — — X X — X — X CMP^(d), LDH X — — X — — X — X Uric acid,Phosphorus X — — X — — X — X Prothrombin time X — — — — — — — — PSA, Ac.Phos X — — X X — X — X Testosterone X — — — — — — — — U/A X — — X X X X— X Stool guaiac X — — — X — — — X Pathology Review^(e) X — — — — — — —— Imaging:^(f) Chest X-ray X — — — — — X — X Bone scan X — — — — — X — XCT Scan or MRI X — — — — — X — X Overall response — — — — — — X — Xassessments^(g) Consent for Pathologic X Correlates^(i) ^(a)Baselinestudies prior to immunization. ^(b)Within 7 days of the firstimmunization. ^(c)Within 15 days of starting treatment for biochemicalstudies; 30 days for imaging studies. Includes total bilirubin, SGOT,LDH, Alkaline Phosphatase, Creatinine, BUN. ^(d)CMP Includes totalbilirubin, SGOT, ALT, Sodium, Potassium, Chloride, CO₂, Calcium,Glucose, Total Protein, Abumin, Alkaline Phosphatase, Creatinine, BUN.^(e)Patients will be asked to obtain tissue blocks from previousdiagnostic/therapeutic procedures will be obtained and the patient'stumor evaluated for the presence of the antigens byimmunohistochemistry. The presence of any antigen on paraffin materialis not a criterion for entry and no biopsy procedures will be performedspecifically for enrollment. ^(f)Abdominal and pelvic CT scans with andwithout contrast, chest x-ray and any other tests deemed necessary todocument evaluable disease. ^(g)Overall response assessment includes therepetition of abnormal imaging and biochemical studies used to assessdisease, and in selected cases, immune function. ^(h)Repeat at 3 monthintervals for 2 years or until disease progression is documented.^(i)Patients will be asked to sign a separate consent for pathologiccorrelative studies under IRB [90-40: Dr. H. Scher, P. I. - Molecularcorrelations in human prostate cancer].

6.3 Immune Function:

STUDY WEEK 0 1 2 3 7 9 13 19 26 VACCINATION* — 1 2 3 4 — — 5 — B-CELLTESTING — 1 2 3 4 5 6 7 8 *No skin tests will be performed as previoustrials indicated that there is minimal or no reactivity with intradermaladministration of the antigens studied.

Antibody Response:

Peripheral blood (30 cc) will be drawn prior to vaccine immediatelybefore each vaccination, as well as weeks 9, 13, and 26 to assess B-cellfunction. Thereafter; blood will be drawn at 3-month intervals (up toone year from the first vaccination), or as long as detectable immunityagainst the antigens persist. Depending on the antibody response,additional testing involving proliferation and cloning may be performedat a later date. The patients' sera will be tested by ELISA forantibodies against purified antigens as well as a variety of cell linesexpressing (or not) the antigens included in the vaccine.

7.0 RESPONSE CRITERIA

7.1 Patients without Bi-Dimensionally Measurable Disease are Evaluableby Post-Therapy Changes in PSA as Follows:

7.1.1 Complete Response (CR):

Normalization of the PSA (≦1.0 or 2.0 as defined in 4.1.1) for 3successive evaluations at least 2 weeks apart.

7.1.2 Partial Response (PR):

Decrease in PSA value by >50% above baseline (without normalization) for3 successive evaluations.

7.1.3 Stabilization (STAB):

Patients who do not meet the criteria for PR or PROG for at least 90days will be considered stable.

7.1.4 Progression (PROG):

Three consecutive increases in PSA, to >50% above baseline.

7.2 Duration of Response:

Non-measurable disease: Time from initiation of therapy until a 50%increase from the PSA nadir value is documented on three successivedeterminations.

8.0 BIOSTATISTICAL CONSIDERATION

8.1 This is an exploratory study to study the safety of a multivalentconjugate vaccine which will be taken to phase III clinical trials.Patients with prostate cancer who have experienced a PSA recurrenceafter radical prostatectomy or radiation therapy are eligible. Allfifteen patients will receive the same dose. The dose is based onprevious vaccine trials in prostate cancer patients, the following doseshave been established for the multivalent trial: glycosylatedMUC-1-32mer, 3 μg; Globo H, 10 μg; GM2, 10 μg; Le^(y), 10 μg; Tn(c), 3μg; and TF(c), 3 μg. QS21 will be used at 100 μg as no significantdifference in immunogenicity was observed with doses as high as 225 μg.Subjects will be followed for two years or until the development ofmetastatic disease. Bone scan and CT scans (or MRI where clinicallyappropriate) will be performed approximately at week 13, 26, andapproximately every 3 months thereafter until the development ofmetastatic disease. In addition, PSA measurements will be obtained atweeks 3, 7, 13, 26, and every approximately 3 months thereafter in orderto study the effect of the vaccine on the probability of developingmetastatic disease and the effect of the vaccine on PSA slope over time,respectively.²⁴8.2 In order to be eligible for the study, patients must have a risingPSA following radical prostatectomy or radiation therapy. This detectionof PSA following treatment must occur within two years.²⁵ sing the ASTROdefinition, three consecutive PSA rises are considered a biochemicalfailure after radical prostatectomy or radiation therapy. The date offailure should be the midpoint between the postsurgical (orpostirradiation) nadir PSA and the first of the three consecutiverises.²⁵ In addition, patients must have a PSA doubling time (DT) lessthan 5 months. PSA doubling time is determined prior to treatment and isequal to ln(2) divided by the least squares derived slope of log PSAover time (log PSA slope>0.15).²⁶ The time interval in which PSA DT willbe based will consist of a minimum of three PSA measurements in atwelve-month interval prior to randomization. Patients who meet thisrequirement are considered at a higher risk for metastatic disease andwill be eligible for this trial.8.3 The primary objective of this study is to determine the safety andthe humoral response of the multivalent vaccine in preparation for thephase III trial. The primary endpoint will be the time to radiographicprogression of disease. The secondary objective is to study the rate ofchange in PSA over time.

REFERENCES

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Gendler S, Lancaster C, Taylor-Papadimitriou J, Duhig T, et al:    Molecular cloning and expression of human tumor-associated    polymorphic epithelial mucin. J Biol Chem 265:15286-15293, 1990.-   8. Gendler S J, Spicer A P, Lalani E-N, et al: Structure and biology    of a carcinoma-associated mucin, MUC1. Am Rev Res, 144:542-S47,    1991.-   9. Carrato C, Balague C, De Bolos C, Gonzalez E, Gambus G, et al:    Differential apomucin expression in normal and neoplastic human    gastrointestinal tissues. Gastroenterology, 107:160-172, 1994.-   10. Ho S B, Niehans G A, Lyftogt C, Yan Ps, et al: Heterogeneity of    mucin gene expression in normal and neoplastic tissues. Cancer Res,    53:641-651, 1993.-   11. Slovin, S. F., Livingston, P., Zhang, S., Keeperman, K., Mhatre,    S., Adluri, S., Ghossein, R., Glazewski, J., Gordils, J.,    Danishefsy, S., and Scher, H. I.: Targeted therapy in prostate    cancer (PC): vaccination with a glycoprotein, MUC-1-KLH-QS21 peptide    conjugate. Proc Am Soc Clin Onc., 16:311a (Abstr 1107), 1997.-   12. Slovin, S. F., Livingston, P., Keeperman, K., Mhatre, S.,    Danishefsky, S., Glazewski, J., Adluri, S., Zhang, S., Gordils, J.,    and Scher, H. I.: Targeted therapy in prostate cancer: vaccination    with glycoprotein, MUC-1-KLH-QS21 peptide conjugate. J. Urol.,    157:160 (Abstr 620), 1997.-   13. Zing P X, Apostolopoulos V, Prenzoska J, et al: Petioled binding    sites recognized by anti-mucin (MUC-2) antibodies. Scand J Immun,    591-592, 1994.-   14. Slovin, S. F., Ragupathi, G., Donaldson, C., Olkiewicz, K.,    Terry, K., DePaolo, R., Livingston, P. O, and Scher, H. I.:    MUC-2-KLH conjugate vaccine: Immunogenicity in patients with    relapsed prostate cancer. Proc. Amer. Assoc. Cancer Res,    40:312(Abstr #2071), 1999.-   15. Ragugpathi, G., Adluri, R., Amaravathi, R., Howarad, L.,    Gilewski, T., Slovin S. F., and Livingston, P. O.: Specificity    analysis of sera from breast and prostate cancer patients vaccinated    with MUC1-KLH and MUC-2-KLH conjugate vaccines. Proc. Amer. Assoc.    Cancer Res, 40:312 (Abstr #2070), 1999-   16. Slovin, S. F., Livingston, P. O., Danishefsky, S., Mhatre, S.,    Ragupathi, R., Depaolo R., Sames, D., Terry K., Bauso, A., Kelly, W.    K., Fazzari, M., and Scher, H. I.: Carbohydrate vaccines as    immunotherapy for prostate cancer (PC): Globo-H-KLH conjugate plus    QS21. Proc Am Soc Clin Onc, 17:433a (Abstr 1669), 1998.-   17. Slovin, S F, Ragupathi G, Adluri S, Ungers G, Terry K, Kim S,    Spassova M, Bornmann, W G, Fazzari M, Dantis L, Olkiewicz K, Lloyd K    O, Livingston P O, Danishefsky S J, and Scher H I: Carbohydrate    vaccines in cancer: Immunogenicity of a fully synthetic globo    hexasaccharide conjugate in man. Proc Natl Acad Sci, USA,    96:5710-5715, 1999.-   18. Slovin, S., Ragupathi, G., Israel, R., Terry, K., Bauso, A.,    Fazzari, M., Kelly, K., Reyes, S., Livingston, P., and Scher, H.    Ganglioside vaccines in relapsed prostate cancer (PC): Experience    with GM2-KLH conjugate plus the immunologic adjuvant, QS21-A trial    comparing QS21 doses. Proc. Amer. Soc. Clin. Onc., 18:316 Aa(Abstr    #1214), 1999.-   19. Livingston P O, Natoli E J, Calves M J: Vaccines containing    purified GM2 ganglioside elicit antibodies in melanoma patients.    Proc natl Acad Sci USA, 27:537, 1987 (Abstract).-   20. Slovin, SF, et al: Tn-cluster (c)-KLH/PAM vaccine conjugates in    biochemically relapsed prostate cancer (PC): Phase I trial results.    Proc. Amer. Assoc. Cancer Res, in press.-   21. MacLean G D, Reddish M, Koganty, R R, Wong T, Gandhi S,    Smolenski M, Samuel J, Nabholtz J M, and Longenecker B M:    Immunization of breast cancer patients using a synthetic sialyl-Tn    glycoconjugate plus Detox adjuvant. Cancer Immunol Immunotherap    36:215-222, 1993.-   22. Zhang S, Walberg L A, Ogata S, Itzkowitz S H, Koganty R R,    Reddish M, Gandhi S S, Longenecker B M, Lloyd K O and Livingston P    O: Immune sera and monoclonal antibodies define two configurations    for the sialyl Tn tumor antigen. Cancer Res 55:3364-3368, 1995.-   23. MacLean G D, Bowen-Yacyshyn M B, Samuel J, Meikle A, Stuart G,    Nation J, Poppema S, Jerry M, Koganty R, Wong T, and Longenecker B    M: Active immunization of human ovarian cancer patients against a    common carcinoma (Thompson-Friedenreich) determinant using a    synthetic carbohydrate antigen. J Immunotherapy, 11:292-305, 1992-   24. Scher, H. I, Slovin, S. F., Kelly, W. K., Livingston, P. O.,    Danishefsky, S., Fazzari, M., Terry, K. And Heller, Glen:    Intermediate markers in assessing response to vaccine therapies.    Proc Am Soc Clin Onc, 17:324a (Abstr 1247), 1998.-   25. Patel, A, Dorey, F, Franklin, J, and DeKernion, J B: Recurrence    patterns after radical retropubic prostatectomy: Clinical usefulness    of prostate specific antigen doubling times and log slope prostate    specific antigen. J Urol, 158:1441-1445, 1997.-   26. O'Brien, PC, Fleming, T R: A multiple testing procedure for    clinical trials. Biometrics 35:549-556, 1979.

Example 2 Hexavalent Vaccine Immunogenicity Trial in Mice MethodsSerological Analyses 1. ELISA (Enzyme-Linked ImmunoSorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2μg/well for glycolipids and 0.1 μg/well for peptides. Serially dilutedantiserum was added to each well and alkaline phosphatase-conjugatedgoat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200(Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goatanti-mouse IgG and IgM conjugated with alkaline phosphatase obtainedfrom Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as secondantibodies. ELISA titer is defined as the highest dilution yielding anabsorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human celllines. Single cell suspensions of 2×10⁵ cells/tube were washed in PBSwith 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with 20 μlof 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice. Aftertwo washes with 3% FCS in PBS, 20 μl of 1:15 diluted goat anti-mouse IgMor IgG-labeled with fluorescein-isothiocyanate (FITC, SouthernBiotechnology Associates Inc. Birmingham, Ala.) was added, and themixture incubated for 30 min. After a final wash, the positivepopulation and mean fluorescence intensity of stained cells weredifferentiated using FACScan, Becton & Dickinson Immunocytometry, SanJose, Calif.

APPENDIX B Hexavalent Vaccine Immunogenicity Trial in Mice Four femaleCB6F1 mice were vaccinated weekly for three weeks with Hexavalentvaccine (Hexavalent vaccine in Polyval-KLH conjugate plus 20 ug QS21 permouse). The injections were SC, at 2 sites, with 95 ul/site. [Viallabeled Polyval in Polyval-KLH conjugate plus 100 ug QS21/1.0 ml. Totalvol. 1.0 ml. Lot # 081100] Pre-vaccination sera was drawn from eachmouse. Sera was drawn again at 10 days post third vaccination. The micewere weighed prior to and post vaccination (at 24 hr post, at 48 hrpost, at one week post and at 2 weeks post). For controls, the followingmonoclonal antibodies were used: VK9 anti-Globo-H BR96 anti-Le^(y) HMFG1anti-MUC1 αTn Ab anti-Tn αGM2 Ab anti-GM2 49H.8 anti-TF SEROLOGY ELISAplates were coated with 0.1 ug/well of one of the following antigens:GloboH-ceramide, GM2(IgM), MUC1G5, Tn-′HSA, Tf-′HSA ELISA plates werecoated with 0.2 ug/well of one of the following antigens: GM2 (IgG), LeySera was tested at an initial dilution of 1:40, with subsequent 2-folddilutions (with the exception of GloboH for which 3-fold dilutions wereused). FACS analysis was performed on two cell lines: MCF7 and LSC (5 ×10⁵ cells per tube). Sera was added at a 1:20 dilution (25 ul/tube).Each post 3rd vacc. sera was set against its corresponding pre-sera(each pre-sera was set at 10%). Appendix B Results ELISA Globo H GM2Le^(Y) IgG IgM IgG IgM IgG IgM Mouse # presera post 3rd presera post 3rdpresera post 3rd presera post 3rd presera post 3rd presera post 3rd 1 040 120 1,080 40 40 40 80 0 40 80 640 2 0 80 40 3,240 40 40 40 80 0 40 8080 4 0 360 120 3,240 0 0 0 80 0 40 40 640 5 0 40 0 3,240 0 40 0 80 0 16040 320 +control VK9 1:25,600 αGM2 >>>1:1,000,000 BR96 (1 ug/ul) 1:3,200MUC1G5 Tf-′HSA Tn-′HSA IgG IgM IgG IgM IgG IgM Mouse # presera post 3rdpresera post 3rd presera post 3rd presera post 3rd presera post 3rdpresera post 3rd 1 0 10,240 0 80 0 640 0 80 0 1,280 0 80 2 0 20,480 0320 0 640 0 160 0 640 0 5,120 4 0  5,120 0 40 0 5,120 0 160 0 10,240 0640 5 0 10,240 0 160 0 10,240 0 160 0 10,240 40 640 +control 49H.81:1,600 aTn 1:25,600 FACS MCF7 LSC IgG IgM IgG IgM Mouse # presera post3rd presera post 3rd +controls presera post 3rd presera post 3rd+controls 1 10.02% 95.76% 11.52% 89.79% VK9 1.13% 10.11% 42.28% 9.63%58.23% VK9 0.95% 2 10.49% 95.30% 10.48% 95.61% BR96 97.11% 9.60% 28.88%10.75% 93.47% BR96 94.85% 4 9.78% 94.71% 11.36% 95.96% HMFG1 62.31%9.93% 27.09% 11.12% 96.28% HMFG1 1.19% 5 9.78% 95.48% 9.86% 94.49% αTnAb 78.62% 10.59% 23.46% 10.16% 93.23% αTn Ab 57.63% 2⁰Ab alone 1.28%1.15% αGM2 Ab 94.91% 1.18% 1.14% αGM2 Ab 63.94% 49H.8 0.14% 49H.8 0.26%

Second Series of Experiments Polyvalent (Heptavalent) Conjugate Vaccinefor Prostate, Breast, Ovarian and Small Cell Lung Cancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewis^(y), GM2,glycosylated MUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safewith local erythema and edema but minimal systemic toxicities. As aresult of vaccination with these monovalent vaccines, most patientsgenerated specific high titer IgM or IgG antibodies against therespective antigen-KLH conjugates. The present invention provides aheptavalent vaccine wherein the components of the monovalent vaccinesare combined and administered with an adjuvant as treatment forprostate, breast, ovarian and small cell lung cancer.

A vaccine consisting of a unique combination of seven tumor antigensadministered with a saponin immunological adjuvant QS-21 or GPI-0100.The antigens are glycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c),sTn(c), and TF(c). In each case the antigen is conjugated to KeyholeLimpet Hemocyanin (KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

Glycosylated MUC-1-G5: 0.1 to 30 μg;

Globo H: 0.1 to 100 μg;

GM2: 0.1 to 100 μg;

Le^(y): 0.1 to 60 μg;

Tn(c): 0.1 to 100 μg;

sTn(c): 0.1 to 100 μg;

TF(c): 0.1 to 30 μg;

QS-21: 25-200 μg;

GPI-0100: 1-25 mg.

Example 1

Phase I clinical trial protocol using the heptavalent vaccine

Example 2

Heptavalent vaccine immunogenicity trial in mice

1. Methods

2. Results

Example 1 Pilot Phase I Trial in Patients with Epithelial Ovarian,Fallopian Tube, or Peritoneal Cancer with a Polyvalent Vaccine-KLHConjugate+Qs-21 1.0 PROTOCOL SUMMARY 2.0 OBJECTIVE 3.0 BACKGROUND ANDRATIONALE 4.0 VACCINE PREPARATION 5.0 TREATMENT SCHEDULE AND DOSE 6.0EVALUATION DURING STUDY 7.0 BIOSTATISTICAL CONSIDERATIONS (Endpoints)8.0 BIBLIOGRAPHY 1.0 PROTOCOL SUMMARY AND PROGRAM PLAN

Patients with epithelial ovarian, fallopian tube, or peritoneal cancerwho receive surgical cytoreduction and platinum/taxane containingchemotherapy have a significant chance of entering complete clinicalremission but unfortunately approximately 70% will eventually relapse.These patients in clinical remission have minimal residual disease, andare excellent candidates in which to evaluate novel consolidationstrategies in an attempt to improve outcome. This pilot polyvalentprotocol represents the culmination of a series of monovalent phase Ivaccine trials at the center demonstrating the immunogenicity of thevarious component antigens. It represents the transition between thephase I monovalent trial program in second remission, to the planneddevelopment of larger trials designed to evaluate efficacy. Immunizationwith the individual antigens selected for this vaccine has beenconsistently immunogenic in the majority of patients. No confirmedsystemic toxicity has occurred related to vaccine administration. It isexpected that the immunogenicity will remain unchanged, and that nosystemic toxicity will occur with polyvalent vaccine administration.Eligible patients for this pilot trial are those patients initially withstage II-IV disease in complete clinical remission following primarytherapy, or following relapse and re-induction to remission withadditional chemotherapy. In this trial, patients will receive an antigendefined vaccine with the following ganglioside components: a) GM2, b)Globo-H; the blood group related antigens: c) TF(c), d) s-Tn (c), e) Tn(c) f) Lewis-Y; and g) the protein antigen MUC-1-G5 (glycosylated). Theprimary endpoints of this pilot study are safety, and confirmation ofcontinued immunogenicity. The secondary endpoint will be to characterizethe nature and duration of the antibody response.

2.0 OBJECTIVES

2.1 The primary endpoints of this pilot study are to determine thesafety of polyvalent vaccine administration, and continuedimmunogenicity in patients prior to conducting a large, randomizedstudy.2.2.2 The secondary endpoint is to further characterize the nature andduration of the antibody response generated by the polyvalent vaccine(ELISA and FACS)

3.0 BACKGROUND AND RATIONALE 3.1 Disease Background and Suitability forTreatment

In 1999, approximately 22,500 new cases of ovarian cancer werediagnosed, and it is estimated that 14,500 women died of the disease.Seventy-five percent of patients with ovarian cancer will have spreadbeyond the ovary at diagnosis. Standard primary treatment consists ofcytoreductive surgery followed by a platinum and paclitaxel containingchemotherapy regimen.¹ Many patients have no clinically measurabledisease at the end of primary treatment. A review of second-looklaparotomy, however, indicates that less than 50% of patients areactually free of disease.² Furthermore, nearly half of patients with anegative second look procedure are destined to relapse and requireadditional treatment.^(3,4) Overall, only approximately 30% of patientsremain disease free with currently available treatment. Given theminimal disease burden at the completion of primary therapy, thesepatients are ideal candidates in which to evaluate immune modulatingstrategies.

3.2 Rationale for Polyvalent Vaccines Designed Primarily for AntibodyProduction

Varied data exists in solid tumors to support the development of immunedirected therapy. Studies have emerged in patients with melanoma whichdemonstrate that naturally acquired ^(5,6), or actively induced ^(7,8)antibodies may improve outcome. In a large clinical trial reported byReithmuller et al., 189 patients with resected Dukes C colon carcinomawere randomized to receive observation versus postoperative treatmentwith murine antibody CO17-1A that recognizes the KSA antigen. Toxiceffects were limited to infrequent constitutional symptoms. At medianfollow-up of five years, the death rate was 36% in the treated groupversus 51% in the observed group. The advantage of treatment wasdemonstrated in univariate (p=0.051) and multivariate (p=0.043) analysiswhen controlling for other known prognostic factors.⁹

The basis for cancer vaccines designed primarily for antibody inductionare the many preclinical models demonstrating the ability of passivelyadministered or actively induced antibodies to prevent tumor recurrencethe increasing number of clinical trials where passively administeredmonoclonal antibodies have demonstrated clinical efficacy, and thecorrelation of antibodies, naturally acquired or vaccine induced, withimproved prognosis in several different clinical settings.⁶

EL4 lymphoma naturally expresses GD2 ganglioside, which is recognized bymonoclonal antibody 3F8. Vaccines containing GD2 covalently conjugatedto KLH and mixed with immunological adjuvant QS21 are the optimalapproach to vaccination against GD2. Relatively higher levels ofantibody administered two or four days after intravenous tumor challengeor moderate titers induced by vaccine that were present by day two orfour after tumor challenge were able to eradicate disease in most mice.If antibody administration was deferred until day seven or ten, littleor no benefit could be demonstrated. If the number of cells in the EL4challenge was decreased, giving a longer window of opportunity, thevaccinations could be initiated after tumor challenge and goodprotection seen.⁷ These results are consistent with the need to initiateimmunization with vaccines inducing antibodies in the adjuvant setting,when the targets are circulating tumor cells and micrometastases.Patients with ovarian cancer in first remission meet these criteria, andunfortunately have a high “event rate” (ie. 80% will relapse) allowingfor the rapid assessment of the efficacy of this approach.

The basis for the inventors' emphasis on polyvalent vaccines is tumorcell heterogeneity, heterogeneity of the human immune response and thecorrelation between overall antibody titer against tumor cells andeffector mechanisms such as complement mediated cytotoxicity (CDC) orantibody dependent cell mediated cytotoxicity (ADCC). For example, usinga series of 14 tumor cell lines and monoclonal antibodies (mAbs) against3 gangliosides, investigators at MSKCC have shown that significant cellsurface reactivity analyzed by flow cytometry and CDC increased from 2-8of the cell lines using one of three mAbs to 13-14 of the cell lineswhen the 3 mAbs were pooled. The median CDC increased 4 fold with thepool of mAbs compared to the best single mAbs.¹¹ Cancers of the ovaryexpress a rich array of cell surface antigens making them especiallysuitable targets for polyvalent vaccines.

Cell surface antigens (especially carbohydrate cell surface antigens)have proven to be unexpectedly potent targets for immune recognition andattack of human cancers. Many of the more tumor-restricted monoclonalantibodies derived by immunization of mice with human tumor cells havebeen found to be directed against carbohydrate antigens expressed at thecell surface¹² Immunization against carbohydrate antigens resultsgenerally in an antibody response (see references for dissenting views),which is primarily IgM.¹³⁻¹⁵ These antibodies are known to induce CDC,inflammation, and phagocytosis of tumor cells by thereticulo-endothelial system (opsonization).¹⁶ Immunization against cellsurface protein antigens can induce a variety of B and T lymphocyteresponses. The T lymphocyte responses are difficult to quantify in thecontext of vaccination trials and are not the focus of this proposal.The antibody responses against protein antigens contain IgM and IgG,both of which can induce complement activation (with regard to IgGdepending on the subclass, IgG1 and IgG3 being optimal). IgG antibodiesof these subclasses can also induce ADCC.

Antibodies are the primary mechanism for active elimination ofcirculating pathogens from the bloodstream. They are ideally suited foreradication of free tumor cells and systemic or intraperitonealmicrometastases and they have accomplished this as described above in avariety of preclinical mouse experiments (reviewed inreferences).^(10,7) In adjuvant immunization trials, the primary targetsare individual tumor cells or early micrometastases which may persistfor long periods after apparent resection of all residual tumor.¹⁷ Aftersurgery and completion of chemotherapy is the ideal time for immuneintervention, and in particular for administration of cancer vaccinesaimed at instructing the immune system to identify and kill the fewremaining cancer cells. If antibodies of sufficient titer can be inducedagainst tumor antigens to eliminate tumor cells from the blood andlymphatic systems, aggressive local therapies, including surgery,radiation therapy and intralesional treatments might result in long termcontrol of even metastatic cancers.

3.3 Preliminary Studies for the Antigens

GM2 vaccines: Investigators at the center have been refining the abilityto induce antibodies against GM2 in melanoma patients for fifteen years,since first demonstrating that vaccines containing purified GM2 could bemore immunogenic than vaccines containing tumor cells expressing GM2.¹⁸Initially GM2 adherent to BCG was selected as optimal, inducing IgMantibodies in 85% of patients. This was the basis for a randomized trialcomparing immunization with BCG to immunization with GM2/BCG in 122patients with AJCC Stage 3 melanoma.⁸ The IgM antibodies had a mediantiter of 1/160 and were short lived (8-12 weeks). IgG antibody inductionwas rare. Antibody titers have been maintained for over three years byadministration of repeated booster immunizations at 3-4 month intervals.When comparing patients as randomized in this trial, no statisticallysignificant difference on overall or disease free survival was seen.Pre-existing GM2 antibodies were seen in 5 patients in the controlgroup, as opposed to one in the GM2 treated group which may have bluntedthe treatment result. The association between better outcome and thepresence of GM2 antibodies was seen (8).

TF, Tn and sTn Vaccines:

Patients with various epithelial cancers have been immunized withunclustered TF-KLH and sTn-KLH conjugate vaccines plus variousadjuvants.¹⁹ High titer IgM and IgG antibodies against TF and sTnantigens have resulted, but we found that the majority of the reactivitydetected in sera from immunized mice and patients was against antigenicepitopes present on synthetic constructs which were not present onnaturally expressed mucins.²⁰ Based on previous studies with Tnantigen,²¹ Kurosaka and Nakada et al. hypothesized that MLS102, amonoclonal antibody against sTn, might preferentially recognize clusters(C) of sTn. In studies at MSKCC with monoclonal antibody B72.3 and withsera raised against TF-KLH and sTn-KLH conjugate vaccines in mice and inpatients resulted in the same conclusion.^(20,22) The availability ofsynthetic TF, Tn and sTn clusters consisting of 3 epitopes covalentlylinked to 3 consecutive serines or threonines has permittedinvestigators at MSKCC to prove this hypothesis. In both direct testsand inhibition assays, B72.3 recognized sTn clusters exclusively, andsera from mice immunized with sTn (C)-KLH reacted strongly with bothnatural mucins and tumor cells expressing sTn.²² Based on thisbackground, we initiated trials with the TF(C)-KLH, Tn(C)-KLH andsTn(C)-KLH conjugate vaccines in patients with breast cancer. Antibodiesof relevant high titer specificity, including against OSM or PSM andcancer cells expressing TF, Tn or sTn, have been induced for the firsttime in the inventors' experience. Based on these results confirming theimportance of clustered epitopes and defining their relevantimmunogenicity, we are including these clustered antigens in thepolyvalent vaccine against ovarian cancer.

Le^(y) and Globo H Vaccines:

The development of Le and Globo H vaccines was previously limited by thelack of sufficient quantities of antigen for vaccine construction andtesting. Over the last four years, Dr. Danishefsky has successfullysynthesized both antigens.^(23,24) Investigators at MSKCC have immunizedgroups of mice with Globo H-ceramide plus or minus adjuvants QS-21 andSalmonella minnesota mutant R595, and with Globo H covalently attachedto KLH or BSA plus immunological adjuvant QS-21. The highest antibodytiters against both synthetic antigen and MCF7 cells expressing Globo Hwere induced by the Globo H-KLH plus QS-21 vaccine.^(23,24) The antibodytiter induced against synthetic Globo H was 1/120,000 by ELISA, thetiter induced against MCF7 was 1/320, and potent complement mediatedcytotoxicity was seen as well. Le^(y)-BSA and Le^(y)-KLH vaccines havealso been tested in the mouse. High titer antibody responses haveresulted against the synthetic epitope of Le^(y) and against tumor cellsexpressing Le^(y) in the majority of mice immunized.²⁵ Based on theseresults, monovalent phase I clinical trials with Globo H-KLH plus QS-21and Le^(y)-KLH plus QS21 have been initiated in patients with breast,prostate or ovary cancer. Antibodies against the purified antigens andagainst tumor cells expressing these antigens were induced in mostpatients and the manuscript was recently published for thelatter.^(26,27)

MUC1 and MUC2 Vaccines:

Investigators at MSKCC have immunized mice with MUC1-KLH and MUC2-KLH,plus QS-21, and seen induction of consistent high titer IgM and IgGantibodies against MUC1 and MUC2 and human cell lines expressing MUC1and MUC2, as well as protection from a syngeneic mouse breast cancerexpressing human MUC1 as a consequence of gene transduction. Mice werealso immunized with vaccines containing MUC1 peptides of various lengthsconjugated to KLH by one of three methods or not, and mixed with QS-21or BCG. MUC1 containing 30 amino acids or more, conjugated to KLH withan MBS bifunctional linker and mixed with immunological adjuvant QS-21induced the highest titer antibodies.²⁸ Based on these studies in themouse, a trial was initiated and completed a trial with this MUC1-KLHplus QS-21 vaccine in breast cancer patients who were free of detectablebreast cancer after resection of all known disease. Nine patients weretreated with a 31 amino acid MUC1 peptide with cysteine at one end forconjugation to KLH and the immune dominant epitope-APDTRPA at the otherend.²⁹ No patient had detectable MUC1 serological reactivity by ELISA orFACS prior to immunization. The results are summarized below in tablebelow. Reactivity against MUC1 and tumor cells expressing MUC1 was seenin most patients. A separate group of patients were immunized withMUC2-KLH plus QS21. Analysis of this trial is not yet complete, but theresults to date are also summarized in the table below.

The inventors have been unable to demonstrate T-lymphocyteproliferation, interferon γ and IL4 release by ELISPOT assays, CTLactivity or positive DTH responses after vaccination with MUC1 or MUC2.The proliferation assays were particularly focussed on in the MUC1trials. Patients had leukophoresis pre and post vaccination, providingample lymphocytes for study. After 2 years of steady endeavor, there hasbeen no clear evidence of augmented reactivity against MUC1 peptides ofvarious lengths or, in HLA A2 positive patients against heteroclyticMUC1 peptides with single amino acid changes that increased binding toHLA A2. (personal communication, P. O. Livingston) Pre and postvaccination PBLs from the first 6 patients vaccinated with MUC1 werealso sent to the laboratory of Dr. Olivera Finn for CTL precursorfrequency analysis. No increase in frequency was seen. Over all, themajor difference between results from MSKCC with the 31aa MUC1-KLH plusQS-21 vaccine and Dr. Finn's results with a 104aa MUC1 peptide plus BCGvaccine was that the former had a clearly demonstrable, consistentantibody response, which was reactive with tumor cells. Inhibitionassays were performed to better understand this serologic response.³⁰Much of the IgM response and nearly all of the IgG response were againstthe immune dominant epitope, APDTRPA, preferentially with RPA at theterminal position.

KSA Vaccines:

KSA has been prepared in the baculovirus system by Jenner Technologies(San Ramon, Calif.) and 10 mcg/patient has been provided for testing.Due to the small quantity of KSA available, following demonstration ofrelevant immunogenicity in mice, we treated groups of 9 patients withKSA plus QS21 or with KSA covalently linked to KLH by glutaraldehyde,plus QS21. In neither case was there significant induction of antibodiesagainst KSA that had not been glutaraldehyde treated, or tumor cellsexpressing KSA. Consequently KSA will not be included in the polyvalentvaccine. The results of this and the other trials with KLH conjugatevaccines are summarized in the table below (personal data, P. O.Livingston).

Summary of Serological Results in Vaccinated Patients Median MedianELISA IgG FACS Median Median Antigen IgM IgG Subclass IgM IgG IA CDC GM2640 320 IgG1 + 3 +++ ++ ++ ++ Globo H 640 40 IgG1 + 3 ++ + ++ +Lewis^(y) 80 0 ++ + + + Tn 1280 1280 ++ − + − STn 1280 160 IgG3 +++ − +− TF 320 10 − + − MUC1 1280 5120 IgG1 + 3 + − + − MUC2 2560 2560 pendingKSA 40 160 − − − −

Additional Variables:

Two additional variables have proven critical, the method of conjugationand the epitope ratio of antigen molecules per KLH molecule. The optimalconjugation approached has varied with the antigen. Gangliosides arebest conjugated using ozone cleavage of the ceramide double bond andintroducing an aldehyde group followed by coupling to aminolysyl groupsof KLH by reductive amination. This approach was not as effective forconjugation of Tn, sTn and TF clusters or Globo H to KLH where an M2C2Hlinker arm has proved most efficient³¹ or for MUC1 or MUC2 where an MBSlinker group was optimal.²⁸

The impact of dose and schedule of vaccine administration on antibodyresponse to GM2 vaccines in melanoma patients has also been explored.Immunization 4 times at weekly intervals or biweekly intervals followedby booster immunizations twice at 2-3 month intervals was compared to 6immunizations at monthly intervals. Initial immunizations at weekly orbiweekly intervals resulted in comparable high titers (with high titersoccurring slightly sooner at weekly intervals), but remarkably themonthly immunizations resulted in far weaker or undetectable antibodyresponses in the 6 patients vaccinated.³² GM2-KLH plus QS-21 vaccinesprepared at MSKCC and at Progenics Pharmaceuticals have each been testedin dose finding studies such as those proposed in this application. Inboth cases GM2 doses of 3 ug or less resulted in lower IgM titers andundetectable IgG titers in most patients. GM2 doses of 10, 30 and 100 uggave comparable IgM and IgG titers.³³ Based an these studies, we haveselected the initial weekly schedule of 3-4 immunizations followed bybooster immunizations every three months, and the doses for use in therandomized Phase III trial.

3.4 Potential Toxicity of Vaccination

The expected safety of the vaccine is based on the safety of vaccinationwith the individual components. Clinical experience is growing inclinical trials with vaccine induced antibody responses against each ofthe included antigens. Antigen expression at secretory borders in thesetrials, where the majority is located, has induced neither immunologicaltolerance nor symptomatic autoimmunity once antibodies are present,suggesting they are sequestered from the immune system. Nevertheless, aregular schedule of laboratory studies and physical examinations aredesigned to detect any abnormalities. This pilot trial will representone of the first studies to confirm the safety of polyvalent vaccineadministration in this setting.

3.5 General Immune Approaches

Various methods have been used to increase the immunogenicity ofantigens, and in particular for inducing an IgG response. In preclinicallaboratory studies, we have found the covalent attachment of antigen tokeyhole limpet hemocyanin (KLH) to be most effective.³⁴ KLH is welltolerated, and has previously caused only mild inflammation at thevaccine injection site. Attachment of KLH may be accomplished by avariety of cross-linking methods. MBS(m-maleimidobenzoly-N-hydroxysuccinimide ester) is the best-knownheterobifunctional reagent; and at neutral pH cross-links thiol groupswith amino groups. The linkage proceeds via two separate reactions, thuslimiting bonds between identical molecules. In addition to linkingantigen to immunogenic carrier proteins, the titer of antibody inducedmay be further augmented with the use of appropriate immunologicaladjuvants. We have immunized groups of melanoma patients with vaccineseither containing no adjuvant, or using DETOX, BCG or QS-21. QS-21 is asignificantly more effective adjuvant than the others, producingsignificantly higher titer IgM and IgG antibodies. It is a saponinderivative extracted from the bark of the South American tree Quillajasaponaria Molina. The monosaccharide composition, molecular weight,adjuvant effect and toxicity for a series of these saponins have beendescribed.³⁵⁻³⁶ It has also proven to be non-toxic and effective ataugmenting the immunogenicity of an FeLV subunit vaccine in cats ³⁷ andan HIV-1 recombinant vaccine in Rhesus monkeys. A phase I trialdemonstrating the safety and suggesting the efficacy of a 100 ug QS-21dose in patients treated with GM2-KLH vaccines has been reported. Theonly adverse events reported were minimal flu-like symptoms, and milddiscomfort at the injection site.³⁸ Thus, conjugation with KLH and theaddition of QS-21 have become standard approaches for vaccineconstruction at MSKCC has proven optimal for antibody induction againsta variety of gangliosides, MUC1, MUC2, KSA, Tn, sTn, TF, Le^(y) andGlobo H in the mouse and in humans.

3.6 Distribution of the Antigens Studied:

In general, the antigens contained in this vaccine are expressed onovarian cancer cells with high frequency. Recent studies at MSKCC havecharacterized this distribution in a variety of tumor types includingovarian cancer using immunohistochemical staining. A variety of tumorspecimens were used in each tumor type, and it was required that 50% ormore cells be positive in order to consider the antigen present. Thepresence of these antigens on the tumor specimens tested in ovariancancer was: GM2 (100%), GLOBO-H (60%), MUC 1 (100%), sTn (60%), TF(100%), Le Y (80%).³⁹⁻⁴¹

3.7 Rational for the Inventors' Approach:

The inventors' hypothesis is that induction of an antibody responseagainst several cell surface antigens on ovarian cancer cells with apolyvalent conjugate vaccine will result in eradication of free tumorantigen, circulating tumor cells and micrometastases. The polyvalentnature of the vaccine and antibody response is important to eliminateescape by tumor cells that fail to express any one or two of theantigens, and to increase the number of antibodies reacting against eachcell. It is expected that the inventors' vaccine will prove consistentlyimmunogenic against five or six of the ovarian cancer cell surfaceantigens described above, and that it will prove nontoxic. Whetherimmunization with this polyvalent vaccine in high-risk ovarian cancerpatients in the remission setting will result in prolonged disease-freeand overall survival is the focus of subsequent studies to follow thispilot trial.

4.0 VACCINE PREPARATION

4.1 GM2 is provided by Progenics. GLOBO-H, Lewis-Y, TF(c), Tn (c), andsTn (c) are synthesized in the laboratory of Dr. Sam Danishefsky at thecenter. MUC-1 is produced at the core facility at MSKCC. Forglycosylation, the MUC-1 was shipped to the University of Copenhagen,Glycobiology Group. The glycosylation was carried out by GalNActransferase using UDP-N-GalNAc as substrate. The product was shippedback to MSKCC after purification by reverse phase HPLC.4.2 QS-21 is obtained from Aquila Biopharmaceuticals in 100 mg vials asa white powder and is stored at −30 degrees Celsius. This is suspendedinitially in PBS as it is less soluble in normal saline and then finaldilutions are made in normal saline. QS-21 is passed through a 0.22micrometer filter immediately prior to use.4.3 Conjugation to KLH is accomplished with three different conjugationprocedures: direct amination (ozonolysis) for GM2, the M2C2Hbifunctional linker group for Globo H and Le Y, and the MBS bifunctionallinker group for the four mucin antigens.4.4 The sterility of the conjugate is confirmed by passage through a0.22 micrometer filter and it is stored in frozen normal saline at −30to −80 degrees Celsius.4.5 Vials are released for use following standard lot release testing(approximately five weeks).

5.0 TREATMENT SCHEDULE AND DOSE 5.1 Vaccine Contains:

[GM2(10 mcg)/TF(c)(3 mcg)/sTn(c) (3 mcg)/Globo-H (10 mcg)/MUC1-1-G5(3mcg)/Le^(Y) (10 mcg)/Tn(c)(3 mcg)]-KLH (≈400 mcg) with adjuvant QS21(100mcg)

5.2 Immunization Schedule

The vaccine will be administered at weekly intervals for 3 doses. Thiswill be followed by a four week break and then a fourth vaccination.There will then be an eight week break and then a fifth vaccination,followed by additional immunizations every twelve weeks for 24 monthstotal (as long as patient remains on study).

IMMUNIZATION SCHEDULE WEEK # 1 2 3 7 15 VACCINE # 1 2 3 4 5

5.3 Dose Administration and Modification

5.3.1 No dose escalation or dose modification will be performed.Systemic toxicity has not been seen with the previous vaccine studies.Systemic toxicity >grade II (with the exception of fever withoutinfection) thought related to vaccination would result in removal of thepatient from study and suspension of the protocol pending investigation.5.3.2 The preparation will be administered subcutaneously to a site inthe shoulders, buttocks, or thighs. It will be administered in onesyringe, and will be supplied in approximately 1 cc total volume.

6.0 EVALUATION DURING STUDY 6.1 Immune Function (Summarized in Table)6.1.1 Antibody Response:

Peripheral blood (20-30 ml) will be drawn according to the schedule intable 8.3 with the exception of week 0, 7, and 9 at which time 50-60 mlwill be collected for antibody testing. Thereafter, blood will be drawnat 12-week intervals as the patients return for booster immunizations.Antibodies against various antigens will be studies by ELISA, andagainst human tumor cell lines by FACS when appropriate.

6.2 Clinical and Laboratory Assessment: 6.2.1 Clinical and LaboratoryAssessment Schedule:

The clinical and laboratory assessment schedule is outlined in Section8.3 and includes parameters to assess the safety of the vaccine, as wellas evaluate for signs of disease recurrence and progression. Abnormalfindings will be evaluated per standard medical practice, and theabnormality will be classified as related to treatment, diseaseprogression, or neither.

6.2.3 Extent of Disease Evaluation:

All patients are by definition without clinical or radiographic evidenceof disease at protocol entry.

6.2.4 Radiographic Imaging

(CT abdomen and pelvis) will be obtained q 6 months while on study, orif any clinical symptoms/examination findings warrant furtherevaluation, or if serum CA-125 rises to ≧70 (per time to treatmentfailure criteria), confirmed by repeat value; or at any time at thediscretion of the investigator.

6.2.5 Length of Follow-Up:

The primary endpoint of the study in this pilot trial is safety.Patients will be followed for the duration of the study, but based onprevious trials, antibodies are generally present by the seventh week,and we will proceed with the proposal for additional studies to evaluateefficacy if no systemic toxicity is seen in any patient at the ninthweek assessment. An additional 8-12 weeks would be required forprocessing before patients could be enrolled on the polyvalent study,allowing ample time for follow-up of the pilot trial. Patients will befollowed until time to treatment failure, or until all vaccinations arecompleted (maximum 24 months).

6.3 Summary of Evaluation:

SUMMARY OF CLINICAL, LABORATORY, AND IMMUNE ASSESSMENTS week # 0^(a) 1 23 5 7 9 13 15 17 27^(c) vaccine # 1 2 3 4 5 V OfficeVisit * * * * * * * * * * * Hx and PE * * * * * * * * *CA-125 * * * * * * CBC, diff * * * * * * * * * * Hepaticprofile + * * * * * * * * * * creatinine Amylase * * * * * * * * * *Urinalysis * * * * * PT * Stool guaiac^(b) * * * * TSH * *Immune^(d) * * * * * * * bloods ^(a)= pretreatment evaluation, within 3weeks ^(b)= stool guaiac may be obtained by digital exam or cardscollected by patient ^(c)= following week 27, visits + laboratorystudies + immunization q 3 months, CT scan q 6 months while on study^(d)= immune bloods routinely consist of 20-30 ml collected in 3 red toptubes. In order to obtain sufficient serum to evaluate for multipleantibodies, 6 tubes will be collected instead of 3 at thepre-vaccination visit; and at week 7 and 9 only.

7.0 BIOSTATISTICAL CONSIDERATIONS Endpoints:

The primary endpoints of this pilot trial are safety and confirmation ofimmunogenicity in the polyvalent setting. No systemic toxicity hasoccurred with the administration of monovalent vaccines at the center.Toxicity is not expected with this preparation. Following this pilot,additional studies with efficacy as the endpoint will be proposed. Thispilot trial would be suspended pending investigation for any systemictoxicity thought related to vaccine in any patient. The same criteriafor immunogenicity will be used as that of the individual pilot trials:patients must have IgM titer >1:80, or a four fold increase inprevailing antibody titer if present at baseline. Nine patients will beaccrued, and >5 of 9 patients should meet these criteria for three ormore antigens in order to proceed with this construct in additionalstudies. In prior trials, antibodies are generally present by completionof the fourth vaccination (week 7). If no systemic toxicity is seen bythe week 9 assessment in these patients, we will proceed with proposalsfor phase II studies with efficacy endpoints.

While not the endpoint of this pilot, patients would be removed fromstudy for relapse as defined below. Time to treatment failure will besimply defined based on data from Rustin et al.⁴² Treatment failure canbe characterized by 1) physical examination evidence of diseaserecurrence, radiographic evidence of disease recurrence (biopsy will beperformed at the discretion of the principal investigator but is notrequired); or 3) CA-125 elevation to twice the upper limits of normal(ie. ≧70), confirmed by a second sample also ≧70 U/ml. Time to treatmentfailure for biochemical relapse is recorded as the date of the firstsample ≧70 U/ml.

The secondary endpoint of this pilot trial is to characterize the natureand duration of the immune response. Peripheral blood (20-30 ml) will bedrawn as indicated in the table. Antibodies against the individualantigens will be studies by ELISA, and against the appropriate humantumor antigen FACS using human tumor cell lines expressing therespective antigen.

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Example 2 Heptavalent Vaccine Immunogenicity Trial 1N Mice MethodsSerological Analyses 1. ELISA (Enzyme-Linked Immunosorbent Assay):

ELISA assays were performed as described below. Antigen in ethanol or in0.1 M carbonate buffer (pH 11) were coated on ELISA plates at 0.2μg/well for glycolipids and 0.1 μg/well for peptides. Serially dilutedantiserum was added to each well and alkaline phosphatase-conjugatedgoat anti-mouse IgM or anti-mouse IgG was added at a dilution of 1:200(Southern Biotechnology Associates, Inc, Birmingham, Ala.). Goatanti-mouse IgG and IgM conjugated with alkaline phosphatase obtainedfrom Kierkegaard and Perry Labs, (Gaithersburg, Md.) were used as secondantibodies. ELISA titer is defined as the highest dilution yielding anabsorbance of 0.1 or greater over that of normal control mouse sera.

2. Cell Surface Reactivity Determined by FACS:

The cell surface reactivity of immune sera was tested on human MCF-7 andLSC cell lines. Single cell suspensions of 2×10⁵ cells/tube were washedin PBS with 3% fetal calf serum (FCS) and 0.01M NaN₃ and incubated with20 μl of 1:20 diluted sera or monoclonal antibody mAb for 30 min on ice.After two washes with 3% FCS in PBS, 20 μl of 1:15 diluted goatanti-mouse IgM or IgG-labeled with fluorescein-isothiocyanate (FITC,Southern Biotechnology Associates Inc. Birmingham, Ala.) was added, andthe mixture incubated for 30 min. After a final wash, the positivepopulation and mean fluorescence intensity of stained cells weredifferentiated using FACScan, Becton & Dickinson Immunocytometry, SanJose, Calif.

Immunizaton of mice with Heptavalent-KLH Conjugates plus ELISA GPI100 OrQS21. ELISA plate mice immunized with . . ./ pre vacc. post vacc. Mar.19, 2001 coated with . . . (Group#) (IgG/IgM) (IgG/IgM) Comments GM2 GM2(group #1) 0/0 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 Muc1-G5(group #4) 0/0 STn(c) (group #5) 0/0 TF(c) (group #6) 0/0 Tn(c) (group#7) 0/0 Globo H GM2 (group #1) GloboH (group#2) 0/0  640/5120 LeY (group#3) Muc1-G5 (group #4) STn(c) (group #5) TF(c) (group #6) Tn(c) (group#7) LeY GM2 (group #1) 0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 640/2560 Muc1-G5 (group #4) 0/0 STn(c) (group #5)  0/160 TF(c) (group#6)  0/40 Tn(c) (group #7)  0/40 Muc1-G5 GM2 (group #1)  0/320 GloboH(group#2)  0/160 LeY (group #3)  0/160 Muc1-G5 (group #4) 0/02560+/1280  STn(c) (group #5) 40/80 TF(c) (group #6) 40/80 Tn(c) (group#7)  0/320 mAb. C595 2560/   B55      /<5120+++ STn(c) GM2 (group #1)0/0 GloboH (group#2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 0/0STn(c) (group #5) 0/0 5120/320  TF(c) (group #6) 0/0 Tn(c) (group #7)0/0 mAb. cc49 1280/0   Tf(c) GM2 (group #1)  0/40 GloboH (group#2) 40/80LeY (group #3) 40/40 Muc1-G5 (group #4) 5120+/160  STn(c) (group #5)5120+/320  TF(c) (group #6) 0/0 5120+/320  Tn(c) (group #7) 5120+/640 mAb. JAA-F11 0/  A78-6/A7   /640 Tn(c) GM2 (group #1) 0/0 GloboH(group#2) 0/0 LeY (group #3) 0/0 Muc1-G5 (group #4) 5120+/160  STn(c)(group #5) 5120+/320  TF(c) (group #6) 5120+/320  Tn(c) (group #7) 0/05120+/2560  mAb. 5F4     /5120++ HB-Tn1   /5120

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21. ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc.Post-vacc Pre-vacc. Post-vacc. Comments 8. Heptavalent-KLH + Tn(c) 1 0/05120+/160 100 ug GPI-100 2 0/0 5120/160 (200 ul/mouse) 3 0/0 5120/160 40/0 5120++/640 5  0/40 5120+/320 Tf(c) 1 0/0 5120/160 2 0/0 5120/160 30/0 5120/160 4 0/0 5120+/320 5  0/40 5120+/640 sTN(c) 1 0/0 5120/640 20/0 1280/640 3 0/0 5120/640 4 0/0 5120/320 5 0/0 1280/320 MUC1-1G5 1 0/05120+/0 2 0/0 5120+/80 3 0/0 5120+++/160 4 0/0 5120++/40 5 0/05120+++/80 Ley 1 0/0   0/40 2 0/0  320/640 3 0/0   0/160 4 0/0  80/40 50/0   0/640 Globo H 1  0/80  40/640 2  0/80   0/640 3  0/80   0/640 4 0/160  40/1280 5  0/160   0/2560 GM2 1 0/0   0/40 2 0/0   0/0 3 0/0  0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent: Tn(c) (3 ug), TF(c) (3 ug),sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10 ug), and GM2(10 ug). New GPI-100 used for all groups except group 9 (batch J).Polysorbate 80, 4 mg/ml in final vaccine. Immunization of mice withHeptavalent-KLH Conjugates* plus GPI-100 or QS-21. Apr. 2, 2001 ELISA(IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc. Post-vaccPre-vacc. Post-vacc. Comments 9. Heptavalent-KLH + Tn(c) 1 0/0  640/40100 ug GPI-100 2 0/0  640/0 (Batch J) 3 0/0 5120/40 (200 ul/mouse) 4 0/05120+/0 5 0/0 1280/40 Tf(c) 1 0/0 1280/80 2 0/0 1280/40 3 0/0 5120+/40 40/0 5120++/40 5 0/0 2560/40 sTN(c) 1 0/0  320/80 2 0/0  320/160 3 0/0 640/160 4 0/0  640/80 5 0/0  80/320 MUC1-1G5 1 0/0 2560/0 2 0/0  640/03 0/0  640/0 4 0/0  640/0 5 0/0 2560/80 Ley 1 0/0   0/0 2 0/0   0/80 30/0   0/0 4 0/0   0/0 5 0/0  160/1280 Globo H 1  0/80   0/160 2  0/80  0/320 3  0/80   0/160 4  0/80   0/320 5  0/80  40/320 GM2 1 0/0   0/02 0/0   0/0 3 0/0   0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent: Tn(c) (3ug), TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H(10 ug), and GM2 (10 ug). New GPI-100 used for all groups except group 9(batch J). Polysorbate 80, 4 mg/ml in final vaccine. Immunization ofmice with Heptavalent-KLH Conjugates* plus GPI-100 or QS-21. Apr. 2,2001 ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc.Post-vacc Pre-vacc. Post-vacc. Comments 10. Heptavalent-KLH + Tn(c) 10/0 5120++/320 100 ug GPI-100 + 2 0/0 5120/320 polysorbate 80 3  0/405120+/1280 (200 ul/mouse) 4 0/0 5120+/640 5 0/0 2560/320 Tf(c) 1 0/05120+/320 2 0/0 5120+/160 3 0/0 5120+/640 4 0/0 5120+/640 5 0/05120+/320 sTN(c) 1 0/0  320/640 2 0/0  320/1280 3 0/0 1280/1280 4 0/0 640/40 5 0/0  320/320 MUC1-1G5 1 0/0 1280/40 2 0/0 5120/80 3 0/05120/160 4 0/0 2560/160 5 0/0 5120/40 Ley 1 0/0   0//0 2 0/0   0/0 3 0/0  0/640 4 0/0   0/640 5 0/0   0/80 Globo H 1  0/40  80/640 2  0/80 40/2560 3  0/320   0/2560 4  0/160   0/2560 5  0/80   0/640 GM2 1 0/0  0/0 2 0/0   0/0 3 0/0   0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent: Tn(c)(3 ug), TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), GloboH (10 ug), and GM2 (10 ug). New GPI-100 used for all groups except group9 (batch J). Polysorbate 80, 4 mg/ml in final vaccine. Immunization ofmice with Heptavalent-KLH Conjugates* plus GPI-100 or QS-21. Apr. 2,2001 ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc.Post-vacc Pre-vacc. Post-vacc. Comments 11. Heptavalent-KLH+ + Tn(c) 10/0 5120/160 10 ug QS-21 2 0/0 5120+/160 (200 ul/mouse) 3  0/405120+++/160 4  0/80 2560/320 5 0/0 5120+/80 Tf(c) 1  0/40 5120+/320 20/0 5120++/640 3  0/160 5120++640 4  0/320 2560/640 5  0/40 5120++/320sTN(c) 1 0/0  40/40 2 0/0 2560/640 3 0/0 5120/160 4 0/0 1280/80 5 0/05120/640 MUC1-1G5 1 0/0 1280/160 2 0/0 2560/80 3 0/0 5120/80 4 0/02560/320 5 0/0 5120++/80 Ley 1 0/0   0//0 2 0/0   0/0 3 0/0   0/0 4 0/0  0/0 5 0/0  40/40 Globo H 1  0/80  40/320 2  0/80  80/320 3  0/80 40/640 4  40/160   0/640 5  0/320   0/2560 GM2 1 0/0   0/0 2 0/0   0/03 0/0   0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent: Tn(c) (3 ug), TF(c) (3ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10 ug), andGM2 (10 ug). New GPI-100 used for all groups except group 9 (batch J).Polysorbate 80, 4 mg/ml in final vaccine. Immunization of mice withHeptavalent-KLH Conjugates* plus GPI-100 or QS-21. Apr. 16, 2001 ELISA(IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice # Pre-vacc. Post-vaccPre-vacc. Post-vacc. Comments 12. Heptavalent-KLH + Tn(c) 1  0/402560/160 3 ug ER-803022 2 0/0  640/160 (200 ul/mouse) 3  0/40  320/320 40/0  160/160 5 0/0 2560/160 Tf(c) 1 0/0 2560/40 2 0/0 2560/40 3 0/0 640/160 4 0/0  320/80 5 0/0 2560/0 sTN(c) 1 0/0   0/40 2 0/0  40/80 340/40  80/160 4 0/0  160/160 5 0/0  40/0 MUC1-1G5 1 0/0  80/0 2 0/0 80/0 3 0/0  80/40 4 0/0  40/80 5 0/0  40/0 Ley 1 0/0   0/0 2 0/0   0/03 0/0   0/0 4 0/0   0/80 5 0/0   0/0 Globo H 1  0/80   0/320 2  0/160 40/1280 3  0/320   0/320 4  80/160  160/1280 5  40/160  320/640 GM2 10/0   0/0 2 0/0   0/0 3 0/0   0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent:Tn(c) (3 ug), TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug),Globo H (10 ug), and GM2 (10 ug). New GPI-100 used for all groups exceptgroup 9 (batch J). Polysorbate 80, 4 mg/ml in final vaccine.Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21. Apr. 16, 2001 ELISA (IgG/IgM) FACS (IgG/IgM) Group # Antigen Mice# Pre-vacc. Post-vacc Pre-vacc. Post-vacc. Comments 13.Heptavalent-KLH + Tn(c) 1  0/40  40/320 10 ug ER-803732 2 0/0  40/40(200 ul/mouse) 3 0/0  320/160 4  0/40  160/160 5  0/40  160/80 Tf(c) 10/0  160/160 2 0/0  320/40 3  0/40  640/160 4  0/40  320/160 5 0/0 320/160 sTN(c) 1 0/0  40/80 2 0/0   0/80 3 0/0   0/320 4  0/160  40/3205  0/40  40/160 MUC1-1G5 1 0/0  640/40 2 0/0   0/40 3 0/0  40/80 4  0/40 80/160 5 0/0  160/80 Ley 1 0/0   0/0 2 0/0   0/0 3  0/40  40/320 4 0/0  0/40 5 0/0   0/80 Globo H 1 40/80  80/160 2  0/160  40/320 3  0/40 80/320 4  0/80   0/160 5  80/160  160/320 GM2 1 0/0   0/0 2 0/0   0/0 30/0   0/0 4 0/0   0/0 5 0/0   0/0 Heptavalent: Tn(c) (3 ug), TF(c) (3ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10 ug), andGM2 (10 ug). New GPI-100 used for all groups except group 9 (batch J).Polysorbate 80, 4 mg/ml in final vaccine. Immunization of mice withHeptavalent-KLH Conjugates* plus GPI-100 or QS-21. ELISA (IgG/IgM) FACS(IgG/IgM) Group # Antigen Mice # Pre-vacc. Post-vacc Pre-vacc.Post-vacc. Comments 14. 30 ug KLH + Tn(c) 1 0/0   0/160 100 ug GPI-100 20/0   0/40 (100 ul/mouse) 3 0/0   0/0 4 0/0   0/160 5  0/40   0/640Tf(c) 1  0/40   0/320 2  0/40   0/160 3 0/0   0/80 4 0/0   0/320 5 0/160  40/640 sTN(c) 1 0/0   0/80 2 0/0   0/40 3 0/0   0/0 4 0/0  0/320 5 0/0   0/80 MUC1-1G5 1 40/40   0/160 2  0/40   0/80 3 0/0  0/80 4 0/0   0/320 5 0/0   0/320 Ley 1 0/0   0/40 2 0/0   0/0 3 0/0  0/0 4 0/0   0/0 5 0/0   0/0 Globo H 1  0/320  40/1280 2  0/320   0/6403  0/80   0/1280 4  0/160   0/640 5  0/160  40/640 GM2 1 0/0   0/160 20/0   0/40 3 0/0   0/0 4 0/0   0/320 5 0/0   0/160 KLH 1 0/0 5120++/3202  0/40 5120++/160 3 0/0 5120++/320 4 0/0 5120++/640 5  0/40 5120++/320

Immunization of mice with Heptavalent-KLH C njugates* plus GPI-100 rQS-21. FACS % positive cells Group # MCF-7 (IgG/IgM) LSC (IgG/IgM) (miceimmunized with . . . ) Mice # Pre-vacc. Post-vacc. 1:200 dilut.Pre-vacc. Post-vacc. 1:200 dilut. 1. 3 ug GM2-KLH + 1 11.79/10.6821.45/11.79 15.73/0.38  10.90/9.58  6.91/2.93 6.56/0.14 100 ug GPI-100 210.23/10.69 4.80/1.49 2.34/0.0  10.90/10.45 5.99/3.45 1.45/0.59 (100ul/mice) 3  9.50/10.74  6.95/15.79 0.99/0.21 11.29/11.11 7.79/1.536.41/0.23 4 9.81/9.68  3.41/12.63 0.99/2.06  9.76/11.06 6.94/1.753.42/0.02 5 10.81/10.42 10.43/3.15  12.29/0.11  10.13/10.76 4.40/1.670.40/0.10 2. 3 ug GloboH-KLH + 1 10.63/9.93   5.79/17.26 6.60/0.19 9.87/10.99 7.08/2.77 9.55/0.25 100 ug GPI100 2 10.08/10.67 16.60/27.6310.54/6.75  10.0/10.41  8.9/3.43 1.29/0.52 (100 ul/mice) 3 11.03/10.0927.21/40.86 27.58/2.30  10.42/10.61 11.56/1.15  7.60/0.02 4 10.16/11.36 1.92/13.77 2.30/1.03 10.57/11.04 5.29/2.43 3.81/0.24 5 11.94/10.8224.61/12.97 20.32/3.62  10.79/10.72 10.58/2.54  1.25/0.20 3. 3 ugLey-KLH + 1 11.01/9.97  85.01/43.57 41.26/1.73  10.22/10.10 89.17/44.2713.40/1.87  100 ug GPI-100 2 9.95/9.69 47.75/78.17 4.86/3.43 10.49/8.39 91.69/79.9  7.64/7.04 (100 ul/mice) 3 10.41/10.58 69.55/10.9520.53/0.12  11.30/9.47  95.57/21.25 24.67/0.16  4 11.93/10.0391.08/0.76  21.29/0.0  10.96/9.60  81.5/6.24 9.08/0.38 5 12.46/11.1842.94/36.67 13.66/1.87  10.98/10.61 12.74/52.16 1.05/1.87 4. 3 ugMuc-1G-5-KLH + 1 11.33/10.94 98.12/9.52  97.98/0.93  9.50/9.89 4.83/1.102.29/0.18 100 ug GPI-100 2 10.90/9.44  88.05/1.47  66.29/0.07 11.39/9.63  8.47/0.15 4.85/0   (100 ul/mice) 3 10.59/10.81 93.34/0.93 83.12/0.09  10.42/9.85  7.42/1.16 3.15/0.02 4 10.47/9.70  94.57/21.9285.52/0.47   9.70/11.11 10.99/7.79  3.47/0.71 5 12.29/10.03 99.77/6.12 99.79/0.77  *11.05/10.49  63.71/1.82  28.90/0.71  5. 3 ug STn(c)-KLH + 19.56/9.77 13.95/1.74  1.29/0.13 10.10/10.73 95.14/4.42  86.94/0.50  100ug GPI-100 2  9.91/10.59 15.95/1.79  12.70/0.23  10.20/11.18 98.65/19.4288.58/0.59  (100 ul/mice) 3 10.72/10.55 6.88/2.81 6.23/0.02 11.31/11.4697.78/31.98 95.31/0.36  4 9.62/9.52 4.62/1.69 3.18/0.14 10.11/9.78 94.14/1.19  86.65/0.09  5 10.77/10.71 17.96/2.16  7.17/0.3  10.71/0.05 97.66/7.28  87.09/0.38  6. 3 ug Tf(c)-KLH + 1 10.84/10.39 13.89/5.73 5.86/0.23  9.64/10.31 7.66/4.74 3.85/0.24 100 ug GPI-100 2  9.22/11.3431.03/4.44  31.75/0.10   9.65/10.15 9.01/1.17 3.98/0.18 (100 ul/mice) 310.59/10.11 4.05/1.05 0.73/0.02 10.12/9.92  4.97/2.77 0.90/0.04 410.26/10.63 6.72/7.10 2.19/0.19  9.72/10.79 3.85/3.58 2.25/0.16 5 12.0/10.90 9.11/6.28 5.18/0.18 *11.54/10.09  11.71/5.0  12.76/0.64  7.3 ug Tn(c)-KLH + 1  9.60/10.44 3.64/8.41 0.62/0.37 10.65/10.46 9.91/5.073.02/0.32 100 ug GPI-100 2 10.54/10.46 12.94/3.24  20.35/0.17 11.50/10.85 4.81/0.48 5.33/0.05 (100 ul/mice) 3 10.51/10.46  4.72/23.220.16/1.22 10.70/9.68  2.37/1.33 0.04/0.02 4 10.97/9.48   8.50/17.774.52/0.16 10.94/10.97 18.50/1.81  4.18/0.12 5 10.79/10.72 46.27/32.779.46/2.04 11.40/10.16 25.42/16.84  16.5/22.80 *DU145 + control IE-391.67, 95.15 +control IE-3 95.73% MLS128 77.07% HMFG1 38.34% MLS13219.92, 4.52  49H8 84.90% 8. Heptavalent-KLH + 1 10.84/11.21 89.44/17.6369.48/0.47  10.29/10.63 97.79/49.78 90.80/0.46  100 ug GPI-100 29.55/9.54 96.98/25.87 83.67/1.12   9.97/10.58 95.44/74.27 83.11/0.35 (200 ul/mice) 3  9.94/10.88 96.53/32.44 88.36/0.79  10.56/10.9195.39/40.29 85.79/0.49  4 10.90/10.72 99.47/5.72  96.61/0.45 10.69/10.42 97.65/16.44 88.77/0.28  5 10.37/10.17  75.64/10.6763.57/0.11  10.58/10.14 93.93/15.49 71.06/1.36  9. Heptavalent-KLH + 110.65/10.57 74.25/12.81 69.56/1.07  10.93/10.21 99.45/8.83  85.59/0.38 100 ug GPI-100 2 10.20/10.97 98.22/16.51 81.61/0.91  10.86/10.7896.70/22.94 66.20/0.50  (batch J) 3 9.96/9.68 89.36/1.03  60.88/0.22 9.79/9.80 95.10/0.43  53.45/0.03  (200 ul/mice) 4 10.77/10.7582.25/8.95  27.80/0.09  10.58/10.28 92.21/1.17  65.13/0.18  510.95/10.77 77.49/10.19 62.39/0.13  10. Heptavalent-KLH + 1 10.99/10.3284.54/8.61  39.91/0.49  10.63/10.92 93.11/38.33 63.61/1.57  100 ugGPI-100 + 2 10.09/10.92 97.83/11.79 94.21/0.16  10.72/10.68 97.64/12.5277.95/0.05  polysorbate 80 3 11.32/10.32 97.74/15.67 89.24/0.30  9.53/10.16 89.84/2.13  63.96/0.19  (200 ul/mice) 4  9.76/10.5992.51/15.57 82.83/0.10  11.50/10.03 60.13/14.56 8.56/1.59 5 10.33/10.0580.12/11.34 70.98/0.30  11. Heptavalent-KLH + 1 10.96/10.08 91.06/1.64 55.98/0.02   9.76/10.81 26.58/2.13  5.42/0.16 10 ug QS-21 2  9.88/10.4194.84/2.32  72.07/0.02   9.80/11.58 88.16/22.37 34.36/0.94  (200ul/mice) 3  11.0/10.57 99.34/9.99  93.96/0.84   9.86/10.79 87.20/11.7076.90/0.14  4 11.05/10.65 88.58/3.78  61.69/0.13  10.07/10.4571.56/3.79  26.42/0.15  5 10.86/10.27 81.44/6.27  69.68/0.14  12.Heptavalent-KLH + 1 18.54/11.29 67.73/1.59  26.33/0.18  10.98/9.97 16.98/1.97  6.40/0.09 3 ug ER803022 2 9.69/9.60 74.76/0.72  33.45/0.08 10.62/10.33 36.76/2.05  3.73/0.07 (200 ul/mice) 3  9.65/10.6291.62/2.89  46.96/0.31  10.16/10.03 28.33/4.88  3.26/0.10 4 10.78/10.0 22.58/4.14  9.51/0.36 5 10.90/10.94 48.15/7.29  10.64/0.23  13.Heptavalent-KLH + 1 10.08/10.56 74.85/8.71  40.57/0.24  10.96/10.2064.39/3.85  7.58/0.17 10 ug ER803732 2 11.03/10.91 36.35/5.07  4.79/0.07 9.84/10.19  3.67/11.73 0.47//0.73 (200 ul/mice) 3 10.22/10.5465.94/30.22 7.87/0.63 10.83/10.07 66.06/25.19 8.66/0.82 4 10.75/10.0791.69/12.15 22.96/0.41  5 10.87/11.04 35.45/6.88  1.38/0.11 14. 30 ugKLH + 1  9.42/11.35 13.72/9.84  10.08/2.29  10.85/10.49 5.49/4.483.29/2.53 100 ug GPI-100 2 12.14/11.31 17.50/2.09  6.41/0.0  10.58/10.4410.57/3.53  14.38/0.21  (200 ul/mice) 3  9.82/10.97 1.64/1.05 0.13/0.02 9.19/11.45 15.10/1.03  11.63/0.01  4 11.20/11.84 13.25/2.14 10.05/0.12  5 10.51/9.95  8.98/5.40 1.38/0.06 Heptavalent: Tn(c) (3 ug),TF(c) (3 ug), sTn(c) (3 ug), MUC1-1G5 (3 ug), Ley (10 ug), Globo H (10ug), and GM2 (10 ug). New GPI-100 used for all groups except group 9(batch J).

Third Series of Experiments Polyvalent Conjugate Vaccine for Cancer

Tumor-specific antigens have been identified and pursued as targets forvaccines. The inventors' previous work has shown that monovalentvaccines utilizing the tumor antigens Globo H, Lewisy, GM2, glycosylatedMUC-1, Tn(c), sTn(c), or TF(c) conjugated to KLH to be safe with localerythema and edema but minimal systemic toxicities. As a result ofvaccination with these monovalent vaccines, most patients generatedspecific high titer IgM or IgG antibodies against the respectiveantigen-KLH conjugates. The present invention provides multivalentvaccines wherein the components of the monovalent vaccine are combinedand administered with an adjuvant as treatment for cancer.

Vaccines consisting of a combination of tumor antigens administered witha saponin immunological adjuvant QS-21 or GPI-0100. The antigens areglycosylated MUC-1-G5, Globo H, GM2, Le^(y), Tn(c), sTn(c), and TF(c).In each case the antigen is conjugated to Keyhole Limpet Hemocyanin(KLH).

The preferred ranges of the antigen and adjuvant doses are as follows:

-   -   Glycosylated MUC-1-G5: 0.1 to 30 g;    -   Globo H: 0.1 to 100 g;    -   GM2: 0.1 to 100 g;    -   Le^(y): 0.1 to 60 g;    -   Tn(c): 0.1 to 100 g;    -   sTn(c): 0.1 to 100 g;    -   TF(c): 0.1 to 30 g;    -   QS-21: 25-200 g;    -   GPI-0100: 1-25 mg.

Example 1 Comparison of the Immune Response after Immunization withMonovalent and Hexavalent-KLH Conjugate Vaccines Against Prostate Cancer

The immune response of the five initial patients receiving hexavalentvaccine with the immune responses of patients who had previously beenimmunized with the respective monovalent vaccines is compared in thefollowing five tables. Shown are the reciprocal mean peak ELISA titerfor IgM and IgG after immunization and FACS assay (% of positivecells/mean intensity) using the MCF-7 tumor cell line. The comparisonfor GM2-KLH is pending. Comparing the responses induced by monovalentand hexavalent vaccines, there was no significant difference in theantibody responses against any of the five antigens tested to date.Combination of six individual conjugates into a single vaccine does notsignificantly change the antibody response against the individualantigens.

Hexavalent Versus Monovalent: Prostate Aug. 6, 2001 MCF-7 FACS PatientElisa (TF-HSA) IgM Trial Patient Sera IgM IgG %/Mean protocol M1 week 1 10 10 10/37 98-048 week 7  1280+ 160 11/39 TF(c)-KLH + week 9 1280 4017/50 QS21 M2 week 1  10 0 10/21 dosage: 1 μg week 7  1280++ 10 52/54week 9   1280+++ 10 72/64 M3 week 1   0 0  11/135 week 7  1280++ 160 2/28 week 9  1280++ 1280  6/104 M4 week 1   0 0 10/32 week 7  160 1018/43 week 9  160 160 26/47 M5 week 1   0 0 10/36 week 7  320 0  9/28week 9  320 0  8/22 protocol H1 week 1  20 0 11/64 00-064 week 7 1280160  7/55 Hexavalent week 12 1280 160  3/31 Conjgate + H2 week 1  40 10 9/37 QS21 week 7 1280 160 21/50 TF(c)dosage: week 12  640 160 17/43 3μg H3 week 1  40 0 10/35 week 7 1280 20 54/65 week 12  640 40 34/47 H4week 1  80 0 10/26 week 7 1280 80 22/43 week 12 1280 20 19/54 H5 week 1  0 0 10/13 week 7 1280 320 57/26 week 12 1280 320 47/22 Controls C1Aug. 27, 1999 1280 1280

Hexavalent Versus Monovalent: Prostate Aug. 10, 2001 MCF-7 Elisa FACSPatient (Tn-HSA) IgM Trial Patient Sera IgM IgG %/Mean protocol 98-002M1 week 1  0 80 10/22 Tn(c)-KLH + QS21 week 7  40 2560  7/18 dosage: 3μg week 9  80 2560  9/21 M2 week 1  10 80 11/26 week 7 320 5120 32/40week 9 640 5120 37/42 M3 week 1  20 40 11/25 week 7 640 640 12/26 week 91280  1280 13/27 M4 week 1  40 40 10/49 week 7 640 1280  9/40 week 91280  1280  8/39 M5 week 1  10 80 11/25 week 7 1280  5120 12/26 week 91280  5120 13/27 protocol 00-064 H1 week 1  80 0 11/64 HexavalentConjgate + week 7 320 1280  7/55 QS21 week 12 320 1280  3/31 Tn dosage:3 μg H2 week 1 320 10  9/37 week 7 1280  320 21/50 week 12 1280  32017/43 H3 week 1  10 0 10/35 week 7 320 640 54/65 week 12 640 640 34/47H4 week 1  40 160 10/26 week 7 640 5120 22/43 week 12 320 5120 19/54 H5week 1  0 20 10/13 week 7 320 640 57/26 week 12 320 640 47/22 ControlsC2 1280+ 640

Hexavalent Versus Monovalent: Prostate Aug. 17, 2001 MCF-7 Elisa FACSPatient (GloboH) IgM Trial Patient Sera IgM IgG %/Mean PROTOCOL 96-055M1 week 1 0 0 11/40 GloboH - KLH + week 7 20 0  9/30 QS21 week 9 20 0 9/40 dosage: 10 μg M2 week 1 0 0 11/33 week 7 0 0 12/42 week 9 0 014/52 M3 week 1 20 0 week 7 640 0 week 9 640 0 M4 week 1 10 0 11/26 week7 160 0 19/39 week 9 160 0 25/39 M5 week 1 40 0 10/26 week 7 160 0 17/38week 9 off study off study PROTOCOL 00-064 H1 week 1 40 0 11/41Hexavalent week 7 160 0  9/36 Conjgate + QS21 week 12 160 80  6/27GloboH dosage: H2 week 1 160 0 11/33 10 μg week 7 640 0 11/36 week 12320 0 15/40 H3 week 1 40 0 11/29 week 7 20 0 56/59 week 12 20 0 43/46 H4week 1 80 0 10/34 week 7 160 0 21/46 week 12 80 0 16/44 H5 week 1 40 010/15 week 7 80 0 64/35 week 12 80 0 45/27 Controls C3 week 26 640 C41280

Hexavalent Versus Monovalent: Prostate Aug. 31, 2001 MCF-7 Elisa FACSPatient Ley-Cer IgM Trial Patient Sera IgM IgG %/Mean PROTOCOL 00-075 M1week 1 0 0 11/23 LewY-MMCCH-KLH week 7 0 0 13/26 dosage: 20 μg week 9 00 24/92 M2 week 1 0 0 10/25 week 7 0 0 20/32 week 9 0 0 14/24 M3 week 10 0 11/30 week 7 40 80  8/20 week 9 20 40 14/41 M4 week 1 0 0 11/46 week7 0 0 10/50 week 9 0 10  9/44 M5 week 1 0 0 10/50 week 7 40 0  3/30 week9 0 10 18/63 PROTOCOL 00-064 H1 week 1 0 0 11/41 Hexavalent Conjgate +week 7 0 0  9/36 QS21 week 12 0 0  6/27 Ley dosage: 10 μg H2 week 1 0 011/33 week 7 0 0 11/36 week 12 0 0 15/40 H3 week 1 0 0 11/29 week 7 10 056/59 week 12 0 0 43/46 H4 week 1 0 10 10/34 week 7 10 10 21/46 week 1210 0 16/44 H5 week 1 0 0 10/15 week 7 0 0 64/35 week 12 0 0 45/27Controls C5 2560 C6 640

Hexavalent Versus Monovalent: Prostate Aug. 19, 2001 MCF-7 Elisa FACSPatient (MUC33G5) IgM Trial Patient Sera IgM IgG %/Mean PROTOCOL 99-23M1 week 1 0 0 10/25 MUC33G 5 Site - week 7 160 640 17/31 KLH + QS21 week9 160 640 31/45 dosage: 3 μg M2 week 1 0 0 10/29 week 7 2560 640 38/53week 9 2560 640 35/43 M3 week 1 0 20 10/28 week 7 2560 160 36/57 week 92560 320 43/60 M4 week 1 0 0 11/41 week 7 2560 80 12/35 week 9 640 32012/38 M5 week 1 0 0 11/30 week 7 40 0 61/65 week 9 40 80 59/67 PROTOCOL00-064 H1 week 1 0 0 11/41 Hexavalent week 7 20 640  9/36 Conjgate +QS21 week 12 0 160  6/27 MUC33 dosage: 3 μg H2 week 1 0 0 11/33 week 7 040 11/36 week 12 0 80 15/40 H3 week 1 0 0 11/29 week 7 160 160 56/59week 12 40 320 43/46 H4 week 1 0 20 10/34 week 7 40 320 21/46 week 12 40160 16/44 H5 week 1 0 0 10/15 week 7 40 160 64/35 week 12 0 80 45/27Controls C7 2560 640

Immunization of mice with Heptavalent-KLH Conjugates* plus GPI-100 orQS-21 Mean Value: Nov. 30, 2001 LSC ELISA FACS MCF-7 # LSC Pre Post PrePost 1:200 Pre Post 1:200 CDC(MCF-7) Group # Antigen IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Mice # Pre Post 1. 3 ugGM2-KLH + Tn 0 0   0 0 10.43% 10.44% 9.40%  8.97% 6.47% 0.55% 10.59%10.60% 6.41% 1.92% 3.63% 0.22% 100 ug GPI-100 Tf (100 ul/mice) sTnMuc1-G5 Ley globo H GM2 2. 3 ug GloboH- Tn 0 0  640 5120 10.77% 10.57%15.22% 22.50% 13.47% 2.78% 10.33% 10.75% 7.08% 2.46% 4.70% 0.25% KLH +Tf 10.08% 10.57% 8.01% 32.59% 6.86% 7.39% 100 ug GPI100 sTn (mice#1 (100ul/mice) Muc1-G5 re- Ley tested.) globo H GM2 3. 3 ug Ley-KLH + Tn 0 0 640 2560 11.15% 10.34% 67.27% 34.02% 20.32% 1.43% 10.79% 9.63% 74.13%40.76% 11.12% 2.26% 3-1  5.94% 46.42% 100 ug GPI-100 Tf 3-2  4.47%75.05% (100 ul/mice) sTn 3-3  4.49%  9.21% Muc1-G5 3-4  22.5% 119.0% Ley3-5 11.02%  61.7% globo H GM2 ELISA FACS MCF-7 LSC Pre Post Pre Post1:200 Pre Post 1:200 CDC(MCF-7) Group # Antigen IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Mice # Pre 4. 3 ug Muc-1G- Tn 00  2560+ 1280 11.12% 10.12% 94.77%  7.99% 86.54% 0.46% 10.25% 10.12%7.93% 2.55% 3.44% 0.23% 4-1  3.30% 5-KLH + Tf 4-2  6.85% 100 ug GPI-100sTn 4-3  14.0% (100 ul/mice) Muc1- 4-4  4.49% G5 4-5  4.23% Ley globo HGM2 5. 3 ug STn(c)-KLH + Tn 0 0 5120 320 10.12% 10.23% 11.88%  2.04%6.11% 0.16% 10.49% 8.64% 96.67% 12.86% 88.91% 0.38% 5-1  6.83% 100 ugGPI-100 Tf 5-2 10.91% (100 ul/mice) sTn 5-3  6.36% Muc1- 5-4  7.11% G55-5  4.37% Ley globo H GM2 6. 3 ug Tf(c)-KLH + Tn 0 0 5120 320 10.58%10.67% 10.56%  4.92% 9.14% 0.14% 9.78% 10.29% 6.37% 3.06% 2.74% 0.16%100 ug GPI-100 Tf (100 ul/mice) sTn Muc1- G5 Ley globo H GM2 7. 3 ugTn(c)-KLH + Tn 0 0  5120+ 2560 10.48% 10.31% 15.21% 17.10% 7.02% 0.79%11.04% 10.42% 12.20% 5.11% 5.81% 4.66% 100 ug GPI-100 Tf (100 ul/mice)sTn Muc1-G5 Ley globo H GM2 KLH LSC ELISA FACS MCF-7 # LSC Pre Post PrePost 1:200 Pre Post 1:200 CDC(MCF-7) Group # Antigen IgG IgM IgG IgM IgGIgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Mice # Pre Post 8.Heptavalent- Tn 0 10  5120+ 320 10.32% 10.5% 91.61% 18.46% 80.34% 0.59%10.43% 10.54% 96.04% 39.25% 83.91% 0.59% 8-1  0.75%  2.99% KLH + Tf 0 105120 320 10.63% 10.14% 98.92% 24.41% 85.27% 7.44% 8-2  1.0% 12.63% 100ug GPI100 sTn 0 0 2560 640 (mice 8-3 −2.23% 44.48% (200 ul/mice) Muc1-G50 0  5120++ 80 #1 8-4 −3.12% 24.77% Ley 0 0  80 160 re- 8-5  7.65%27.28% globo H 0 80  10 1280 tested) VK9  2.05% GM2 0 0   0 10 3S193 82.2% anti-GM2  71.2% 696  32.2% ELISA FACS MCF-7 LSC Pre Post Pre Post1:200 Pre Post 1:200 CDC(MCF-7) Group # Antigen IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG IgM IgG IgM IgG IgM Mice # Pre Post 9. Heptavalent-Tn 0 0 2560 40 10.51% 10.55% 84.31%  9.89% 60.45% 0.48% 10.40% 10.21%92.22% 16.68% 63.24% 0.25% 9-1 −2.79% 34.02% KLH + Tf 0 0 5120 40 9-2 2.96%  9.42% 100 ug GPI (old sTn 0 0  320 160 9-3  4.50% 11.41% batch)Muc1-G5 0 0 1280 0 9-4  6.74%  9.51% (200 ul/mice) Ley 0 0  40 320 9-5 4.01% 39.91% globo H 0 80   0 320 3S193 97.83% GM2 0 0   0 0 B72.3 5.34% HMFG.1  3.01% 10. Heptavalent- Tn 0 0  5120+ 640 10.50% 10.44%90.55% 12.59% 75.43% 0.27% 10.67% 10.24% 85.22% 11.17% 54.16% 0.74% 10-1−6.29%  −3.3% KLH + Tf 0 0 5120 320 100 ug GPI-100 + sTn 0 0  640 640polysorbate 80 Muc1-G5 0 0 5120 80 (200 ul/mice) Ley 0 0   0 320 globo H0 160  20 1280 GM2 0 0   0 0 11. Heptavalent- Tn 0 20  5120+ 160 10.75%10.39% 90.96%  4.8% 70.67% 0.23% 9.92% 10.74% 70.28% 8.33% 40.71% 0.29%11-1  1.84% 10.93% KLH + Tf 0 80  5120+ 640 11-2  0.71%  3.20% 10 ugQS-21 sTn 0 0 2560 320 11-3  1.31%  5.88% (200 ul/mice) Muc1-G5 0 0 2560160 11-4 −1.58% 18.36% Ley 0 0  10 10 11-5 10.28% 27.09% globo H 0 80 40 640 GM2 0 0   0 0 12. Heptavalent- Tn 0 0 1280 160 11.89% 10.49%60.97%  3.33% 25.38% 0.23% 10.42% 10.29% 29.79% 4.55% 5.65% 0.14% KLH +Tf 0 0 1280 80 3 ug ER803022 sTn 0 0  80 80 (200 ul/mice) Muc1-G5 0 0 80 20 Ley 0 0   0 20 globo H 20 160  80 640 GM2 0 0   0 0 13.Heptavalent- Tn 0 20  160 160 10.59% 10.62% 60.86% 12.60% 15.51% 0.29%10.50% 10.24% 45.30% 11.86% 5.60% 0.60% KLH + Tf 0 10  320 160 10 ugER803732 sTn 0 40  20 160 (200 ul/mice) Muc1-G5 0 0  160 80 Ley 0 0   080 globo H 20 80  80 320 GM2 0 0   0 0 14. 30 ug KLH + Tn 0 0   0 16010.62% 11.08% 11.02%  4.10% 5.61% 0.45% 10.32% 10.68% 9.09% 2.47% 7.96%0.73% 100 ug GPI-100 Tf 0 40   0 320 (100 ul/mice) sTn 0 0   0 80Muc1-G5 0 10   0 160 Ley 0 0   0 0 globo H 0 160  10 640 GM2 0 0   0 160KLH 0 10  5120++ 320

Immunization of mice with Heptavalent-KLH Conjugates* plus QS-21 or GPI100 with or without mAb 9H10 against CTLA-4. Mean Value: ELISA (mean)ELISA (median) FACS MCF-7

Pre Post Pre Post Pre Post 1:200

Group # Antigen IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgM IgG IgMAVERAGE MEDIAN 1. Heptavalent-KLH + 10 ug QS21 Tn 0 0 57440  640 0 025600  640 9.89% 9.78% 95.87% 77.77% 78.03% 16.09% +CONTROL Tf 0 0 10240++ 260 0 0 10240+ 160 9.39% 9.94% 96.40% 80.96% 79.70% 8.16% VK-940.08% sTn 0 0 3520 580 0 0 1280 320 MLS128 97.56% Muc1-G5 0 0 1280 80 00 1280 80 antiGM2 #### Ley 0 32 1920 2560 0 0 1280 1280 MBr-1 #### globoH 0 48  672 128 0 80  640 160 GM2 2. Heptavalent-KLH + 100 ug Tn 0 019200  840 0 0 25600  320 10.15% 10.36% 92.97% 79.87% 66.63% 28.35%GPI100 Tf 0 0 10240+ 260 0 0 10240+ 160 10.10% 10.35% 95.99% 93.53%69.88% 26.96% sTn 0 0  720 360 0 0  320 80 Muc1-G5 0 0 1520 80 0 0 2560160 Ley 0 0 3920 680 0 0 5120 640 globo H 0 0  320 176 0 0  80 160 GM23. Heptavalent-KLH + 100 ug GPI- Tn 0 0 32000  480 0 0 25600  320 10.29%10.14% 92.85% 62.94% 65.36% 18.34% 100 (Lyophilized) Tf 0 0 10240+ 340 00 10240+ 320 10.20% 10.23% 92.35% 58.56% 63.59% 14.46% sTn 0 0 1200 2600 0 1280 320 Muc1-G5 0 0 1120 0 0 0  320 0 Ley 0 0  320 720 0 0  480 640globo H 0 0  128 440 0 0   0 200 GM2 4. Heptavalent-KLH + 100 ug Tn 0 038400  400 0 0 25600  160 10.14% 10.18% 91.34% 57.09% 70.79% 6.70%GPI100′ + polysorbate80 Tf 0 0 10240+ 140 0 0 10240+ 160 10.22% 10.29%90.80% 61.70% 67.27% 4.98% sTn 0 0 1960 200 0 0 1280 320 Muc1-G5 0 04000 20 0 0 2560 0 Ley 0 0 1440 960 0 0 1280 640 globo H 0 40  100 224 040  120 160 GM2 5. Heptavalent-KLH + 100 ug GPI100 Tn 0 0 61400  808 0 025600  640 9.92% 10.30% 89.55% 72.17% 64.38% 12.43% Cytoxan 25 mg/Kg(I.P.) Day −1 Tf 0 0  10240++ 560 0 0  10240++ 640 10.19% 10.19% 90.87%70.90% 66.42% 14.32% sTn 0 0  520 480 0 0  640 320 Muc1-G5 0 0 3840 0 00 2560 0 Ley 0 0  480 200 0 0   0 80 globo H 0 0  32 540 0 0   0 400 6.Heptavalent-KLH + 100 ug GPI100 Tn 0 0 32000  250 0 0 25600  160 10.33%10.08% 90.59% 65.89% 62.40% 17.62% mAb CTLA-4 in vaccine Tf 0 0 10240+1040 0 0 10240+ 1280 10.26% 10.01% 91.50% 62.97% 63.04% 8.00% (100ug/mice) Day 0 sTn 0 0 6240 180 0 0 1280 200 Day 7& 14 no CTLA-4 Muc1-G50 0 1950 40 0 0 2560 40 Ley 0 0  180 2600 0 0  40 40 globo H 0 0   0 2400 0   0 240 GM2 7. Heptavalent-KLH + 100 ug GPI100 Tn 0 0 32000  1600 00 25600  1280 10.05% 10.31% 90.31% 85.42% 67.29% 29.21% mAb CTLA-4 invaccine Tf 0 0 10240+ 1920 0 0 10240+ 1280 9.64% 10.41% 90.01% 85.58%69.08% 21.26% (100 ug/mice) Day 0 & 7 sTn 0 0 2880 100 0 0 2560 80 Day14 no CTLA-4 Muc1-G5 0 0 6400 60 0 0 5120 80 Ley 0 0  32 208 0 0   0 160globo H 0 0  32 220 0 0   0 240 GM2 8. Heptavalent-KLH + 100 ug GPI100Tn 0 0 85333  40 0 0 51200+ 40 9.69% 10.15% 91.29% 45.33% 63.00% 6.19%mAb CTLA-4 not in vaccine Tf 0 0  10240++ 370 0 0  10240++ 320 9.63%10.09% 90.20% 45.21% 50.05% 7.00% I.P. day −1, 0, 1 sTn 0 0  480 0 0 0 640 0 Muc1-G5 0 0 3413 0 0 0 2560 0 Ley 0 0  320 106 0 0  160 0 globo H0 0  53 240 0 0   0 80 GM2 9. Heptavalent-KLH + 100 ug GPI100 Tn 0 056320  640 0 0 51200  640 10.25% 10.13% 93.16% 90.64% 73.15% 45.88%Control mAb ROR-g2 Tf 0 0  10240+++ 2720 0 0  10240+++ 2560 10.27%10.21% 94.17% 95.54% 76.62% 30.38% 100 ug/mice sTn 0 0 3040 5200 0 01280 320 I.P. day −1, 0, 1 Muc1-G5 0 0 10240  160 0 0 10240  160 Ley 0 0 144 464 0 0  80 160 globo H 0 0  112 848 0 0  80 640 GM2 10.Heptavalent-KLH + 100 ug GPI100 Tn 0 0 81920  320 0 0 51200  160 10.42%9.98% 88.03% 76.96% 67.02% 33.26% 100 ug/mice Tf 0 0  10240++ 920 0 0 10240++ 320 10.50% 10.01% 87.97% 90.19% 64.58% 24.15% sTn 0 0 2240 14400 0 2560 160 Muc1-G5 0 0 4160 40 0 0 2560 0 Ley 0 144  144 2640 0 160 80 320 globo H 0 0   0 224 0 0   0 160 GM2

Example 2 A Preclinical Study Comparing Approaches for Augmenting theImmunogenicity of a Heptavalent KLH-Conjugate Vaccine Against EpithelialCancers

Previously using a series of monovalent vaccines, we have demonstratedthat the optimal method for inducing an antibody response against cancercell-surface antigens is covalent conjugation of the antigens to keyholelimpet hemocyanin (KLH) and the use of a saponin adjuvant. Inpreparation for testing a polyvalent (heptavalent)-KLH conjugate vaccinein the clinic, we have tested the impact on antibody induction againstthe 7 antigens of several variables described by others to augmentimmunogenicity. We explore here the impact of approaches for decreasingsuppression of the immune response (low dose cyclophosphamide andanti-CTLA4 mAb), different saponin adjuvants (QS-21 and GPI-0100), anddifferent methods of formulation (lyophilization and use of polysorbate80). After two sets of experiments, these results are clear:

1) Immunization with the heptavalent-KLH conjugate vaccine induces hightiters of antibodies against Tn (median ELISA titer IgM/IgG 320/10,240),sTn (640/2560), TF (320/5120), MUC1 (80/20,480) and globo H (1280/10),lower titers of antibodies against Lewis Y (160/80) and only occasionalantibodies against GM2.

2) These antibodies reacted with the purified synthetic antigens byELISA, and with naturally expressed antigens on the cancer cell surfaceby FACS.3) Neither decreasing suppression with low dose cyclophosphamide oranti-CTLA4 mAb, nor changing the standard formulation by lyophilizationor use of polysorbate 80 had any impact on antibody titers.4) The two saponin adjuvants were comparably potent at our standarddoses (QS-21 bug and GPI-0100 100 ug) but a third experiment comparinghigher doses is in progress.

The high titers of antibodies against this heptavalent vaccine and theinability of these additional approaches to further augment antibodytiters confirms that the combination of conjugation to KLH and use of asaponin adjuvant is sufficiently optimized for testing in the clinic.

There is a broad and expanding body of pre-clinical and clinical studiesdemonstrating that naturally acquired, actively induced, and passivelyadministered antibodies are able to eliminate circulating tumor cellsand micro metastases (1). Induction of antibodies against tumor antigensis more difficult than induction of antibodies against viral andbacterial antigens because most tumor antigens are normal or slightlymodified auto antigens and because actively growing tumors may set inmotion mechanisms which suppress the anti-cancer cell immune response.Consequently it may be necessary to overcome not only some level oftolerance but also some additional level of active suppression, makingthe immunization approach critical. We have previously reported that theoptimal approach for induction of antibodies against gangliosides and avariety of other carbohydrate and peptide antigens is covalentattachment of the tumor antigen to an immunogenic carrier molecule(keyhole limpet hemocyanin (KLH) was optimal (2,3)) plus the use of apotent immunological adjuvant. In our previous experience saponinadjuvants such as QS-21 and GPI-0100 were the optimal adjuvants (4,5).

In preparation for clinical trials with a heptavalent KLH-conjugatevaccine we test here the impact of several variables including 1)vaccine formulation (lyophilization or the use of polysorbate 80), 2)decreasing suppression (low dose cyclophosphamide or anti-CTLA4 mAb), or3) various doses of the two saponin adjuvants QS-21 and GPI-0100, onantibody titers against the individual antigens and tumor cellsexpressing these antigens.

Pathogen-free female BALB/c or C57BL/6 mice 6-10 weeks of age wereobtained from the Jackson Laboratory (Bar Harbor, Me.). QS-21 wasobtained from Aquila Biopharmaceuticals (Framingham, Mass. (nowAntigenics Inc., NYC, NY)), GPI-0100 was obtained from GalenicaPharmaceuticals, Inc. (Birmingham, Ala.). Cytoxan (25 mg/kg) waspurchased and injected IP one day prior to the first immunization. Thehybridoma for murine monoclonal antibody CTLA-4 was obtained from JimAllison (Berkeley, Calif.) and the mAb was prepared by Dr. Polly Gregor(MSKCC). The reactivity of mAb with CTLA-4 was confirmed. Polysorbate 80was purchased.

Immunization of Mice:

Groups of five mice were immunized 3 times at one week intervals withthe heptavalent vaccine containing 3 mcg of each of the 7 antigenscovalently conjugated to KLH and mixed with GPI-0100 or QS-21 asindicated. Vaccines were administered subcutaneously over the lowerabdomen. A 4th, booster, immunization was given at week 8.

Serological Assays:

For the ELISA assay, glycosylated MUC1, globo H, Lewis Y or GM2, or Tn,sTn or TF conjugated to BSA, were coated on ELISA plates at an antigendose of 0.1-0.2 mcg per well.

Phosphatase-conjugated goat anti-mouse IgG or IgM was added at adilution of 1:200 (Southern Biotechnology Associates, Inc., Birmingham,Ala.). Antibody titer was the highest dilution yielding absorbance of0.10 or greater.

F ACS Analysis:

MCF-7 human breast cancer cells expressing all seven antigens butespecially Lewis Y and MUC1 and sTn, and LSC expressing especially LewisY, sTn and Tn, were used. Single cell suspensions of 5×10⁷ cells/tubewere washed in PBS with 3% fetal calf serum and incubated with 20 mcl offull strength or 1/200 diluted antisera for 30 minutes on ice. 20microliters of 1/15 goat anti-mouse IgG or IgM labeled with FITC wereall added and percent positive cells and mean fluorescent intensity(MFI) of stained cells analyzed using a F ACScan (Becton Dickenson,Calif.). Pre and post vaccination sera were analyzed together and thepre-treatment percent positive cells set at 10%.

Comparison of the Immune Response after Immunization with Monovalent andHexavalent-KLH Conjugate Vaccines Against Prostate Cancer

Glycolipid and glycoprotein differentiation antigens such as GM2, GloboH, Lewis y, Tn, TF, and mucin 1 (MUC1) are over-expressed on the cellsurface of many tumors. Of the many approaches to immunization we havetested, covalent conjugation of antigens such as these to keyhole limpethemocyanin (KLH) plus the use of immunological adjuvant QS-21 has beenthe optimal approach for inducing IgM and IgG antibodies. Immunizationof patients with monovalent vaccines containing these antigens hasdemonstrated the consistent immunogenicity and safety of these vaccines.However, to overcome the heterogeneous nature of tumors, and of theimmune response in different individuals, we have recently vaccinated asmall group of patients (prostate cancer with rising PSA, but free ofdetectable disease) with a hexavalent-KLH vaccine containing GM2, GloboH, Le^(y), Tn(c), TF(c) and glycosylated MUC1 individually conjugatedwith KLH and mixed with immunological adjuvant QS-21. The main objectiveof this presentation is to compare the immune response of the sixinitial patients receiving hexavalent vaccine with the immune responsesof patients who had previously been immunized with the respectivemonovalent vaccines. All patients were vaccinated six times (weeks1,2,3,7,19 and 31) and bloods obtained pre treatment and on weeks 7 and9 were tested at one time. RECIPRICOL MEAN PEAK ELISA TITER AFTERIMMUNIZATION IgM/IgG.

Antigen Globo GM2 H Le^(y) Tn TF MUC1 Polyvalent Pending 160/0 0/0 640/640 1280/160  40/320 vaccine Individual Pending 160/0 0/10 1280/25601280/160 2560/320 Vaccine

Because of the low response against Le^(y), we are continuing studiesaimed at creating a more immunogenic Le^(y) vaccine. Comparing theresponses induced by monovalent and hexavalent vaccines, there was nosignificant difference in the antibody responses against any of the fiveantigens tested to date. Combination of six individual conjugates into asingle vaccine does not significantly change the antibody responseagainst the individual antigens.

Experiment 1: Median ELISA titers and FACS results after vaccination ofgroups of 5 Balb/c mice with Heptavalent-KLH conjugate ELISA(mean)ELISA(median) Pre Post Pre Post Group # Antigen IgG IgM IgG IgM IgG IgMIgG IgM 1. Heptavalent-KLH + Tn 0 0 57440  640 0 0 25600  640 10 ug QS21Tf 0 0  10240++ 260 0 0 10240+ 160 sTn 0 0 3520 580 0 0 1280 320 Muc1-G50 0 1280 80 0 0 1280 80 Ley 0 32 1920 2560 0 0 1280 1280 globo H 0 48 672 128 0 80  640 160 GM2 2. Heptavalent-KLH + Tn 0 0 19200  840 0 025600  320 100 ug GPI100 Tf 0 0 10240+ 260 0 0 10240+ 160 sTn 0 0  720360 0 0  320 80 Muc1-G5 0 0 1520 80 0 0 2560 160 Ley 0 0 3920 680 0 05120 640 globo H 0 0  320 176 0 0  80 160 GM2 3. Heptavalent-KLH + Tn 00 32000  480 0 0 25600  320 100 ug GPI-100 Tf 0 0 10240+ 340 0 0 10240+320 (Lyophilized) sTn 0 0 1200 260 0 0 1280 320 Muc1-G5 0 0 1120 0 0 0 320 0 Ley 0 0  320 720 0 0  480 640 globo H 0 0  128 440 0 0   0 200GM2 4. Heptavalent-KLH + Tn 0 0 38400  400 0 0 25600  160 100 ugGPI100′ + Tf 0 0 10240+ 140 0 0 10240+ 160 polysorbate80 sTn 0 0 1960200 0 0 1280 320 Muc1-G5 0 0 4000 20 0 0 2560 0 Ley 0 0 1440 960 0 01280 640 globo H 0 40  100 224 0 40  120 160 GM2 5. Heptavalent-KLH + Tn0 0 61400  808 0 0 25600  640 100 ug GPI100 Tf 0 0  10240++ 560 0 0 10240++ 640 Cytoxan 25 mg/Kg sTn 0 0  520 480 0 0  640 320 (I.P.) Day−1 Muc1-G5 0 0 3840 0 0 0 2560 0 Ley 0 0  480 200 0 0   0 80 globo H 0 0 32 540 0 0   0 400 GM2 6. Heptavalent-KLH + Tn 0 0 32000  250 0 025600  160 100 ug GPI100 Tf 0 0 10240+ 1040 0 0 10240+ 1280 mAb CTLA-4in sTn 0 0 6240 180 0 0 1280 200 vaccine Muc1-G5 0 0 1950 40 0 0 2560 40(100 ug/mice) Day 0 Ley 0 0  180 2600 0 0  40 40 Day 7& 14 no globo H 00   0 240 0 0   0 240 CTLA-4 GM2 7. Heptavalent-KLH + Tn 0 0 32000  16000 0 25600  1280 100 ug GPI100 Tf 0 0 10240+ 1920 0 0 10240+ 1280 mAbCTLA-4 in sTn 0 0 2880 100 0 0 2560 80 vaccine Muc1-G5 0 0 6400 60 0 05120 80 (100 ug/mice) Day 0 Ley 0 0  32 208 0 0   0 160 & 7 globo H 0 0 32 220 0 0   0 240 Day 14 no CTLA-4 GM2 8. Heptavalent-KLH + Tn 0 085333  40 0 0 51200+ 40 100 ug GPI100 Tf 0 0  10240++ 370 0 0  10240++320 mAb CTLA-4 not in sTn 0 0  480 0 0 0  640 0 vaccine Muc1-G5 0 0 34130 0 0 2560 0 I.P. day −1, 0, 1 Ley 0 0  320 106 0 0  160 0 globo H 0 0 53 240 0 0   0 80 GM2 Pre Post Pre Post 9. Heptavalent-KLH + Tn 0 056320  640 0 0 51200  640 100 ug GPI100 Tf 0 0  10240+++ 2720 0 0 10240+++ 2560 Control mAb ROR-g2 sTn 0 0 3040 5200 0 0 1280 320 100ug/mice Muc1-G5 0 0 10240  160 0 0 10240  160 I.P. day −1, 0, 1 Ley 0 0 144 464 0 0  80 160 globo H 0 0  112 848 0 0  80 640 GM2 10.Heptavalent-KLH + Tn 0 0 81920  320 0 0 51200  160 100 ug GPI100 Tf 0 0 10240++ 920 0 0  10240++ 320 100 ug/mice sTn 0 0 2240 1440 0 0 2560 160Muc1-G5 0 0 4160 40 0 0 2560 0 Ley 0 144  144 2640 0 160  80 320 globo H0 0   0 224 0 0   0 160 GM2 FACS MCF-7 Pre Post 1:200 Group # AntigenIgG IgM IgG IgM IgG IgM +CONTROL 1. Heptavalent-KLH + Tn 10% 10% 96% 78%78% 16% VK-9 40.08% 10 ug QS21 Tf  9% 10% 96% 81% 80%  8% MLS128 97.56%sTn antiGM2 98.87% Muc1-G5 MBr-1 58.06% Ley globo H GM2 2.Heptavalent-KLH + Tn 10% 10% 93% 80% 67% 28% 100 ug GPI100 Tf 10% 10%96% 94% 70% 27% sTn Muc1-G5 Ley globo H GM2 3. Heptavalent-KLH + Tn 10%10% 93% 63% 65% 18% 100 ug GPI-100 Tf 10% 10% 92% 59% 64% 14%(Lyophilized) sTn Muc1-G5 Ley globo H GM2 4. Heptavalent-KLH + Tn 10%10% 91% 57% 71%  7% 100 ug GPI100′ + Tf 10% 10% 91% 62% 67%  5%polysorbate80 sTn Muc1-G5 Ley globo H GM2 5. Heptavalent-KLH + Tn 10%10% 90% 72% 64% 12% 100 ug GPI100 Tf 10% 10% 91% 71% 66% 14% Cytoxan 25mg/Kg sTn (I.P.) Day −1 Muc1-G5 Ley globo H GM2 6. Heptavalent-KLH + Tn10% 10% 91% 66% 62% 18% 100 ug GPI100 Tf 10% 10% 92% 63% 63%  8% mAbCTLA-4 in sTn vaccine Muc1-G5 (100 ug/mice) Day 0 Ley Day 7& 14 no globoH CTLA-4 GM2 7. Heptavalent-KLH + Tn 10% 10% 90% 85% 67% 29% 100 ugGPI100 Tf 10% 10% 90% 86% 69% 21% mAb CTLA-4 in sTn vaccine Muc1-G5 (100ug/mice) Day 0 Ley & 7 globo H Day 14 no CTLA-4 GM2 8. Heptavalent-KLH +Tn 10% 10% 91% 45% 63%  6% 100 ug GPI100 Tf 10% 10% 90% 45% 50%  7% mAbCTLA-4 not in sTn vaccine Muc1-G5 I.P. day −1, 0, 1 Ley globo H GM2 9.Heptavalent-KLH + Tn 10% 10% 93% 91% 73% 46% 100 ug GPI100 Tf 10% 10%94% 96% 77% 30% Control mAb ROR-g2 sTn 100 ug/mice Muc1-G5 I.P. day −1,0, 1 Ley globo H GM2 10. Heptavalent-KLH + Tn 10% 10% 88% 77% 67% 33%100 ug GPI100 Tf 11% 10% 88% 90% 65% 24% 100 ug/mice sTn Muc1-G5 Leyglobo H GM2

Experiment 2: Median ELISA titers and FACS results against MCF-7 and LSCcells after vaccination groups of 5 C57BL/6 Mice with heptavalent-KLHconjugate vaccine FACS ELISA withMCF-7 Pre Post Pre Post Group # AntigenIgG IgM IgG IgM IgG IgM IgG IgM Heptavalent-KLH + Tn 0 10  5120+ 320 10%11% 91.61% 18.46% 100 ug GPI100 Tf 0 10 5120 320   99%   24% (200ul/mice) sTn 0 0 2560 640 Muc1-G5 0 0  5120++ 80 Ley 0 0  80 160 globo H0 80  10 1280 GM2 0 0   0 10 Heptavalent-KLH + Tn 0 0 2560 40 11% 11%84.31%  9.89% 100 ug GPI (old Tf 0 0 5120 40 batch) sTn 0 0  320 160(200 ul/mice) Muc1-G5 0 0 1280 0 Ley 0 0  40 320 globo H 0 80   0 320GM2 0 0   0 0 Heptavalent-KLH + Tn 0 0  5120+ 640 11% 10% 90.55% 12.59%100 ug GPI-100 + Tf 0 0 5120 320 polysorbate 80 sTn 0 0  640 640 (200ul/mice) Muc1-G5 0 0 5120 80 Ley 0 0   0 320 globo H 0 160  20 1280 GM20 0   0 0 Heptavalent-KLH + Tn 0 20  5120+ 160 11% 10% 90.96%  4.8% 10ug QS-21 Tf 0 80  5120+ 640 (200 ul/mice) sTn 0 0 2560 320 Muc1-G5 0 02560 160 Ley 0 0  10 10 globo H 0 80  40 640 GM2 0 0   0 0Heptavalent-KLH + Tn 0 20  160 160 11% 11% 60.86% 12.60% 10 ug Tf 0 10 320 160 ER803732 sTn 0 40  20 160 200 ul/mice) Muc1-G5 0 0  160 80 Ley0 0   0 80 globo H 20 80  80 320 GM2 0 0   0 0 FACS FACS with LSCwithMCF-7 # LSC 1:200 Pre Post 1:200 Group # Antigen IgG IgM IgG IgM IgGIgM IgG IgM Heptavalent-KLH + Tn 80% 18% 10% 11% 96.04% 39.25% 84% 39%100 ug GPI100 Tf 82% 21% (200 ul/mice) sTn Muc1-G5 Ley globo H GM2Heptavalent-KLH + Tn 60% 10% 10% 10% 92.22% 16.68% 63% 17% 100 ug GPI(old Tf batch) sTn (200 ul/mice) Muc1-G5 Ley globo H GM2Heptavalent-KLH + Tn 75% 13% 11% 10% 85.22% 11.17% 54% 11% 100 ugGPI-100 + Tf polysorbate 80 sTn (200 ul/mice) Muc1-G5 Ley globo H GM2Heptavalent-KLH + Tn 71%  5% 10% 11% 70.28%  8.33% 41%  8% 10 ug QS-21Tf (200 ul/mice) sTn Muc1-G5 Ley globo H GM2 Heptavalent-KLH + Tn 16%13% 11% 10% 45.30% 11.86%  6% 12% 10 ug Tf ER803732 sTn 200 ul/mice)Muc1-G5 Ley globo H GM2

Pr

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col 00-106: Pilot Phas

 Trial: Vaccination of Patients Who Have Ovarian, Fallopian Tube orPeritoneal Cancer with A Polyvalent Vaccine-KLH Conjugate + QS-21Patient Name Vaccine: Pa- 10 ug GM2 10 ug Globo-H 10 ug LeY tientMuc1-1G5 Globo-H LeY # Vaccination Serology Sera # IgM IgG IgM IgG IgMIgG Jun. 12, 2002 Jun. 12, 2002 Jun. 27, 2002 Jun. 19, 2002 Jun. 21,2002 Jun. 21, 2002 Pa- Jul. 03, 2001 Jul. 03, 2001 P7OVQ 0 0 0 0 0 0tient 2 1 Jul. 10, 2001 Jul. 31, 2001 P7OVQ 2560 640 40 0 0 0 6 Jul. 17,2001 Aug. 14, 2001 P7OVQ 2560 640 40 0 0 0 9 Aug. 14, 2001 Aug. 08, 2001P7OVQ 2560 320 40 0 0 0 15 OFF TRIAL (+) Control M62 2560 640 Positve2560 control S193(1 6400 mg/ml) Positve 2560 control Positve controlPositve control Positve control Positve 2560 control Jun. 04, 2002 Jun.04, 2002 Jun. 27, 2002 Jun. 26, 2002 Pa- Jul. 03, 2001 Jul. 03, 2001P7OVQ 0 0 0 0 tient 3 2 Jul. 10, 2001 Jul. 31, 2001 P7OVQ 80 20 0 0 7Jul. 17, 2001 Aug. 14, 2001 P7OVQ 40 20 0 0 10 Aug. 14, 2001 Aug. 28,2001 P7OVQ 20 40 0 0 13 Sep. 25, 2001 P7OVQ 20 20 0 0 20 Oct. 09, 2001Oct. 09, 2001 P7OVQ 20 20 0 0 24 Oct. 23, 2001 P7OVQ 20 20 0 0 30 Jan.11, 2002 P7OVQ 0 0 0 0 49 Jun. 07, 2002 P7OVQ 0 0 65 (+) Control Positve2560 640 control Positve 2560 control Positve 640 control Patient NameVaccine: Pa- 3 ug Muc1G5 3 ug Tn(c) 3 ug S-Tn(c) 3 ug TF(c) tient GM2Tn(c) S-Tn(c) TF(c) # Vaccination Serology Sera # IgM IgG IgM IgG IgMIgG IgM IgG Jun. 24, 2002 Jun. 24, 2002 Jun. 25, 2002 Jun. 25, 2002 Pa-Jul. 03, 2001 Jul. 03, 2001 P7OVQ 0 0 0 20 tient 2 1 Jul. 10, 2001 Jul.31, 2001 P7OVQ 0 20 1280 160 6 Jul. 17, 2001 Aug. 14, 2001 P7OVQ 0 201280 160 9 Aug. 14, 2001 Aug. 09, 2001 P7OVQ 0 20 1280 320 15 OFF TRIAL(+) Control M62 Positve control S193(1 mg/ml) Positve control Positve640 control Positve 2560 control Positve 2560 2560 control Positvecontrol Pa- Jul. 03, 2001 Jul. 03, 2001 P7OVQ tient 3 2 Jul. 10, 2001Jul. 31, 2001 P7OVQ 7 Jul. 17, 2001 Aug. 14, 2001 P7OVQ 10 Aug. 14, 2001Aug. 28, 2001 P7OVQ 13 Sep. 25, 2001 P7OVQ 20 Oct. 09, 2001 Oct. 09,2001 P7OVQ 24 Oct. 23, 2001 P7OVQ 30 Jan. 11, 2002 P7OVQ 49 Jun. 07,2002 P7OVQ 65 (+) Control Positve control Positve control Positvecontrol Patient Name Med- ical Muc1-1G5 Globo-H LeY # VaccinationSerology Sera # IgM IgG IgM IgG IgM IgG Jun. 07, 2002 Jun. 07, 2002 Jun.27, 2002 Jun. 26, 2002 Pa- Jul. 10, 2001 Jul. 10, 2001 P7OVQ 0 0 20 0tient 4 3 Jul. 17, 2001 Aug. 07, 2001 P7OVQ 1280 160 160 0 8 Jul. 24,2001 Aug. 21, 2001 P7OVQ 320 40 80 0 12 Aug. 21, 2001 Sep. 04, 2001P7OVQ 160 80 80 0 18 Oct. 02, 2001 P7OVQ 80 40 40 0 22 Oct. 16, 2001Oct. 16, 2001 P7OVQ 40 40 80 0 26 Oct. 30, 2001 P7OVQ 80 80 40 0 33 Jan.08, 2002 P7OVQ 40 40 80 0 47 (+) Control M62 2560 1280 Positve 2560control Positve 1280 control Jun. 10, 2002 Jun. 10, 2002 pa- Jul. 24,2001 Jul. 24, 2001 P7OVQ 0 0 tient 5 4 Jul. 31, 2001 Aug. 21, 2001 P7OVQ1280 80 14 Aug. 07, 2001 Sep. 04, 2001 P7OVQ 1280 40 17 Sep. 04, 2001Sep. 18, 2001 P7OVQ 160 20 23 Oct. 16, 2001 P7OVQ 160 20 27 Oct. 30,2001 Oct. 30, 2001 P7OVQ 80 40 32 Nov. 13, 2001 P7OVQ 20 20 36 Jan. 22,2002 P7OVQ 20 20 52 Mar. 26, 2002 P7OVQ 20 20 63 (+) Control M62 1280640 Jun. 12, 02 Jun. 12, 02 Jun. 27, 2002 Jun. 19, 2002 Jun. 21, 2002Jun. 21, 2002 pa- Aug. 16, 2001 Aug. 16, 2001 P7OVQ 0 0 0 0 0 0 tient 115 Aug. 23, 2001 Sep. 27, 2001 P7OVQ 80 80 0 0 0 0 21 Aug. 30, 2001 Oct.11, 2001 P7OVQ 40 80 0 0 0 0 25 Sep. 27, 2001 OFF TRIAL (+) Control M622560 640 Positve 2560 control S193(1 6400 mg/ml) Positve 2560 controlPositve control Positve control Positve control Positve 2560 controlJun. 17, 2002 Jun. 17, 2002 Pa- Aug. 30, 2001 Aug. 30, 2001 P7OVQ 0 0tient 16 6 Sep. 06, 2001 Sep. 27, 2001 P7OVQ 160 20 19 Sep. 13, 2001Oct. 25, 2001 P7OVQ 40 20 31 Oct. 11, 2001 Nov. 22, 2001 P7OVQ 20 0 39Dec. 06, 2001 Dec. 06, 2001 P7OVQ 20 0 41 Dec. 20, 2001 P7OVQ 20 20 44Mar. 08, 2002 P7OVQ 20 20 62 (+) Control M62 2560 320 Jun. 13, 2002 Jun.13, 2002 Jun. 27, 2002 Jun. 19, 2002 Jun. 21, 2002 Jun. 21, 2002 pa-Oct. 19, 2001 Oct. 19, 2001 P7OVQ 0 0 0 (re-do) 0 0 tient 28 7 Oct. 26,2001 Nov. 16, 2001 P7OVQ 640 40 0 (re-do) 0 0 35 Nov. 02, 2001 Dec. 14,2001 P7OVQ 80 20 0 (re-do) 0 0 42 Nov. 30, 2001 Jan. 11, 2002 P7OVQ 8020 0 (re-do) 0 0 48 Jan. 25, 2002 Jan. 25, 2002 P7OVQ 80 20 0 (re-do) 00 53 Feb. 08, 2002 P7OVQ 80 20 0 (re-do) 0 0 56 Apr. 19, 2002 (+)Control M62 2560 160 Positve 2560 control S193(1 6400 mg/ml) Positve2560 control Positve control Positve control Positve control Positve2560 control Jun. 18, 2002 Jun. 18, 2002 Pa- Oct. 23, 2001 Oct. 23, 2001P7OVQ 0 0 tient 29 8 Oct. 30, 2001 Nov. 20, 2001 P7OVQ 0 0 38 Nov. 06,2001 Dec. 18, 2001 P7OVQ 0 0 43 Dec. 04, 2001 Jan. 15, 2002 P7OVQ 0 0 51Jan. 29, 2002 Jan. 29, 2002 P7OVQ 40 0 54 OFF TRIAL (+) Control M62 2560640 Jun. 18, 2002 Jun. 18, 2002 Pa- Nov. 06, 2001 Nov. 06, 2001 P7OVQ 00 tient 34 9 Nov. 13, 2001 Dec. 04, 2001 P7OVQ 1280 80 40 Nov. 20, 2001Jan. 01, 2002 P7OVQ 320 80 45 Dec. 18, 2001 Jan. 29, 2002 P7OVQ 320 8055 Feb. 12, 2002 Feb. 12, 2002 P7OVQ 160 40 58 Feb. 26, 2002 P7OVQ 16080 60 May 09, 2002 P7OVQ 40 80 64 (+) Control M62 2560 320 Jun. 13, 2002Jun. 13, 2002 Jun. 27, 2002 Jun. 19, 2002 Jun. 21, 2002 Jun. 21, 2002Pa- Nov. 20, 2001 Nov. 20, 2001 P7OVQ 0 0 0 (re-do) 0 0 tient 37 10 Nov.27, 2001 Dec. 18, 2001 P7OVQ 320 80 320 (re-do) 0 20 46 Dec. 04, 2001Jan. 15, 2002 P7OVQ 320 320 320 (re-do) 0 20 50 Jan. 01, 2002 Feb. 12,2002 pt no — — — — — — show Feb. 26, 2002 Feb. 26, 2002 P7OVQ 40 160 80(re-do) 0 20 59 Mar. 12, 2002 pt no — — — — — — show May 21, 2002 pt no— — — — — — show (+) Control M62 2560 160 Positve 2560 control S193(16400 mg/ml) Positve 2560 control Positve control Positve control Positvecontrol Positve 1280 control Jun. 12, 2002 Jun. 12, 2002 Jun. 27, 2002Jun. 19, 2002 Jun. 21, 2002 Jun. 21, 2002 Pa- Mar. 07, 2001 Mar. 07,2001 P7OVQ 0 0 0 0 0 0 tient 1 11 Feb. 05, 2002 Feb. 05, 2001 P7OVQ 0 00 0 0 0 57 Feb. 12, 2002 Mar. 05, 2002 P7OVQ 0 20 0 0 0 0 61 Feb. 19,2002 OFF TRIAL Mar. 19, 2002 (+) Control M62 2560 640 Positve 2560control S193(1 6400 mg/ml) Positve 2560 control Positve control Positvecontrol Positve control Positve 2560 control Patient Name Med- ical GM2Tn(c) STn(c) Tf # Vaccination Serology Sera # IgM IgG IgM IgG IgM IgGIgM IgG Pa- Jul. 10, 2001 Jul. 10, 2001 P7OVQ tient 4 3 Jul. 17, 2001Aug. 07, 2001 P7OVQ 8 Jul. 24, 2001 Aug. 21, 2001 P7OVQ 12 Aug. 21, 2001Aug. 04, 2001 P7OVQ 18 Oct. 02, 2001 P7OVQ 22 Oct. 16, 2001 Oct. 16,2001 P7OVQ 26 Oct. 30, 2001 P7OVQ 33 Jan. 08, 2002 P7OVQ 47 (+) ControlM62 Positve control Positve control pa- Jul. 24, 2001 Jul. 24, 2001P7OVQ tient 5 4 Jul. 31, 2001 Aug. 21, 2001 P7OVQ 14 Aug. 07, 2001 Sep.04, 2001 P7OVQ 17 Sep. 04, 2001 Aug. 18, 2001 P7OVQ 23 Oct. 16, 2001P7OVQ 27 Oct. 30, 2001 Oct. 30, 2001 P7OVQ 32 Nov. 13, 2001 P7OVQ 36Jan. 22, 2002 P7OVQ 52 Mar. 26, 2002 P7OVQ 63 (+) Control M62 Jun. 24,2002 Jun. 24, 2002 Jun. 25, 2002 Jun. 25, 2002 pa- Aug. 16, 2001 Aug.16, 2001 P7OVQ 0 0 0 0 tient 11 5 Aug. 23, 2001 Sep. 27, 2001 P7OVQ 0 040 160 21 Aug. 30, 2001 Oct. 11, 2001 P7OVQ 0 0 0 160 25 Sep. 27, 2001OFF TRIAL (+) Control M62 Positve control S193(1 mg/ml) Positve controlPositve 640 control Positve 2560 control Positve 2560 2560 controlPositve control Pa- Aug. 30, 2001 Aug. 30, 2001 P7OVQ tient 16 6 Sep.06, 2001 Sep. 27, 2001 P7OVQ 19 Sep. 13, 2001 Oct. 25, 2001 P7OVQ 31Oct. 11, 2001 Nov. 22, 2001 P7OVQ 39 Dec. 06, 2001 Dec. 06, 2001 P7OVQ41 Dec. 20, 2001 P7OVQ 44 Mar. 08, 2002 P7OVQ 62 (+) Control M62 Jun.24, 2002 Jun. 24, 2002 Jun. 25, 2002 Jun. 25, 2002 pa- Oct. 19, 2001Oct. 19, 2001 P7OVQ 0 0 20 0 tient 28 7 Oct. 26, 2001 Nov. 16, 2001P7OVQ 0 0 160 160 35 Nov. 02, 2001 Dec. 14, 2001 P7OVQ 0 0 80 160 42Nov. 30, 2001 Jan. 11, 2002 P7OVQ 0 20 80 80 48 Jan. 25, 2002 Jan. 25,2002 P7OVQ 0 20 80 40 53 Feb. 08, 2002 P7OVQ 0 20 80 80 56 Apr. 19, 2002(+) Control M62 Positve control S193(1 mg/ml) Positve control Positve640 control Positve 2560 control Positve 2560 2560 control Positvecontrol Pa- Oct. 23, 2001 Oct. 23, 2001 P7OVQ tient 29 8 Oct. 30, 2001Nov. 20, 2001 P7OVQ 38 Nov. 06, 2001 Dec. 18, 2001 P7OVQ 43 Dec. 04,2001 Jan. 15, 2002 P7OVQ 51 Jan. 29, 2002 Jan. 29, 2002 P7OVQ 54 OFFTRIAL (+) Control M62 Pa- Nov. 06, 2001 Nov. 06, 2001 P7OVQ tient 34 9Nov. 13, 2001 Dec. 04, 2001 P7OVQ 40 Nov. 20, 2001 Jan. 01, 2002 P7OVQ45 Dec. 18, 2001 Jan. 29, 2002 P7OVQ 55 Feb. 12, 2002 Feb. 12, 2002P7OVQ 58 Feb. 26, 2002 P7OVQ 60 May 09, 2002 P7OVQ 64 (+) Control M62Jun. 24, 2002 Jun. 24, 2002 Jun. 25, 2002 Jun. 25, 2002 Pa- Nov. 20,2001 Nov. 20, 2001 P7OVQ 0 0 0 0 tient 37 10 Nov. 27, 2001 Dec. 18, 2001P7OVQ 0 20 160 80 46 Dec. 04, 2001 Jan. 15, 2002 P7OVQ 0 20 80 640 50Jan. 01, 2002 Feb. 12, 2002 pt no — — — — show Feb. 26, 2002 Feb. 26,2002 P7OVQ 0 20 40 320 59 Mar. 12, 2002 pt no — — — — show May 21, 2002pt no — — — — show (+) Control M62 Positve control S193(1 mg/ml) Positvecontrol Positve 640 control Positve 2560 control Positve 2560 2560control Positve control Jun. 24, 2002 Jun. 24, 2002 Jun. 25, 2002 Jun.25, 2002 Pa- Mar. 07, 2001 Mar. 07, 2001 P7OVQ 0 20 20 0 tient 1 11 Feb.05, 2002 Feb. 05, 2001 P7OVQ 0 0 20 0 57 Feb. 12, 2002 Mar. 05, 2002P7OVQ 0 0 40 80 61 Feb. 19, 2002 OFF TRIAL Mar. 19, 2002 (+) Control M62Positve control S193(1 mg/ml) Positve control Positve 640 controlPositve 2560 control Positve 2560 2560 control Positve control * Patientreceived one vaccine before protocol hold, restarted ~1 year later

indicates data missing or illegible when filed

Protocol # 01-019: Serological analysis of Breast cancer patientvaccinated with hexavalent vaccine ELISA GM2 ELISA MUC-1-1 ELISA LeYCeramide ELISA Globo H (10 mcg)(March 5G (3 (10 mcg) Ceramide(10 VaccineSample 2002) mcg)(March 2002) (April 2002) mcg) (May 2002) Patient #Date Date Serology Top IgG IgM IgG IgM IgG IgM IgG Patient Jan. 16, 2002Jan. 16, 2002 P7BRQ4 20 0 0 0 0 0 0 1 Jan. 23, 2002 Jan. 23, 2002 P7BRQ90 0 0 0 0 0 0 Jan. 30, 2002 Jan. 30, 2002 P7BRQ11 0 0 0 0 0 0 0 Feb. 13,2002 P7BRQ19 0 0 40 80 0 0 0 Feb. 27, 2002 Feb. 27, 2002 P7BRQ28 0 0 2080 0 0 0 Mar. 13, 2002 P7BRQ37 0 40 40 80 0 0 0 Apr. 10, 2002 P7BRQ44 80320 0 0 0 May 22, 2002 May 22, 2002 P7BRQ62 Jun. 05, 2002 P7BRQ67Controls >2560 320 (M62) 320 2560 (MCG170) 160 2560 (LeYM12) 20 80/160Mono Ab (S193) 1280 (GB81) 0 Mono Ab (VK9) 5120 (P7BRQ17) (slovin lab wk7) Patient Jan. 16, 2002 Jan. 16, 2002 P7BRQ5 0 0 0 0 0 0 0 2 Jan. 23,2002 Jan. 23, 2002 P7BRQ8 0 0 0 0 0 0 0 Jan. 30, 2002 Jan. 30, 2002P7BRQ12 0 0 0 0 0 0 0 Feb. 13, 2002 P7BRQ20 0 0 80 160 0 0 0 Feb. 27,2002 Feb. 27, 2002 P7BRQ27 0 80 40 20 0 0 0 Mar. 13, 2002 P7BRQ36 0 16080 20 0 0 0 Apr. 10, 2002 P7BRQ46 80 80 0 0 0 May 22, 2002 May 22, 2002P7BRQ61 Jun. 05, 2002 P7BRQ68 Controls >2560 320 (P7BRQ17) >2560(hexavalent) 80 320 (P7BRQ16) 640 (LeYM12) 0 160 Mono Ab (S193) 1280(GB81) 0 Mono Ab (VK9) 5120 (P7BRQ17) (slovin lab wk 7) Patient Jan. 07,2002 Jan. 07, 2002 P7BRQ1 0 0 0 0 0 0 0 3 Jan. 14, 2002 Jan. 14, 2002P7BRQ3 0 0 0 0 0 0 0 Jan. 21, 2002 Jan. 21, 2002 P7BRQ7 0 0 0 320 0 20 0Feb. 04, 2002 P7BRQ17 0 20 80 2560 0 40 0 Feb. 18, 2002 Feb. 18, 2002P7BRQ23 0 0 80 640 0 20 0 Mar. 04, 2002 P7BRQ30 0 0 80 320 0 0 0 Apr.01, 2002 P7BRQ41 80 80 0 0 0 May 13, 2002 May 13, 2002 P7BRQ57 0 May 27,2002 P7BRQ63 0 Controls >2560 (P7BRQ17) 320 (hexavalent) 160 1280(P7BRQ16) 320 2560 (LeYM12) 20 160 Mono Ab (S193) 1280 (GB81) 0 Mono Ab(VK9) 0 (P7BRQ17) (slovin lab wk 7) Patient Jan. 14, 2002 Jan. 14, 2002P7BRQ2 0 0 0 0 0 40 0 4 Jan. 21, 2002 Jan. 21, 2002 P7BRQ6 0 0 0 0 0 2020 Jan. 28, 2002 Jan. 28, 2002 P7BRQ10 0 0 0 80 0 40 0 Feb. 11, 2002P7BRQ16 0 0 640 320 0 1280 0 Feb. 25, 2002 Feb. 25, 2002 P7BRQ26 0 0 16040 0 320 0 Mar. 11, 2002 P7BRQ34 0 0 160 20 0 640 80 did not Apr. 08,2002 come in Apr. 15, 2002 P7BRQ47 320 80 0 320 0 May 20, 2002 May 20,2002 P7BRQ58 0 Jun. 03, 2002 P7BRQ66 80 Controls >2560 (P7BRQ17) 320(hexavalent) 320 >2560 (P7BRQ16) 160 >2560 (LeYM12) 20 160 Mono Ab(S193) 1280 (GB81) 20 Mono Ab (VK9) 0 (P7BRQ17) (slovin lab wk 7)Patient Feb. 06, 2002 Feb. 06, 2002 P7BRQ15 0 0 0 0 0 0 0 5 Feb. 13,2002 Feb. 13, 2002 P7BRQ22 0 0 0 20 0 0 0 Feb. 20, 2002 Feb. 20, 2002P7BRQ25 0 0 0 0 0 0 0 Mar. 06, 2002 P7BRQ33 0 0 40 40 0 0 0 Mar. 20,2002 Mar. 20, 2002 P7BRQ38 0 0 20 40 0 0 0 Apr. 03, 2002 P7BRQ42 160 400 0 0 May 01, 2002 P7BRQ51 0 Jun. 12, 2002 Jun. 12, 2002 P7BRQ71 Jun.26, 2002 Controls >2560 (P7BRQ17) 320 80 160 (hexavalent) 640 320(P7BRQ16) 20 160 (LeYM12) 1280 Mono Ab (S193) 0 (GB81) 0 Mono Ab (VK9)(P7BRQ17) (slovin lab wk 7) Patient Feb. 06, 2002 Feb. 06, 2002 P7BRQ140 20 0 0 20 0 0 6 Feb. 13, 2002 Feb. 13, 2002 P7BRQ18 0 20 0 0 0 0 0Feb. 20, 2002 Feb. 20, 2002 P7BRQ24 0 40 0 80 0 0 0 Mar. 06, 2002P7BRQ31 0 20 320 320 0 0 0 Mar. 20, 2002 Mar. 20, 2002 P7BRQ39 0 20 160320 0 0 0 Apr. 03, 2002 P7BRQ43 320 40 0 0 0 May 01, 2002 P7BRQ52 0 Jun.12, 2002 Jun. 12, 2002 P7BRQ70 Jun. 26, 2002 Controls >2560 320(P7BRQ17) 80 640 (hexavalent) 640 320 (P7BRQ16) 0 80/160 (LeYM12) 1280Mono Ab (S193) 0 (GB81) 0 Mono Ab (VK9) (P7BRQ17) (slovin lab wk 7)ELISA Globo H ELISA Tf(3 ELISA dOSM ELISA Ceramide(10 mcg)(May for Tn (3mcg) sTn Vaccine Sample mcg) (May 2002) 2002) (June 2002) (3 mcg)Patient # Date Date Serology Top IgM IgG IgM IgG IgM IgG IgM PatientJan. 16, 2002 Jan. 16, 2002 P7BRQ4 0 0 20 1 Jan. 23, 2002 Jan. 23, 2002P7BRQ9 0 0 40 Jan. 30, 2002 Jan. 30, 2002 P7BRQ11 20 0 40 Feb. 13, 2002P7BRQ19 20 320 640 Feb. 27, 2002 Feb. 27, 2002 P7BRQ28 0 80 1280 Mar.13, 2002 P7BRQ37 20 160 1280 Apr. 10, 2002 P7BRQ44 20 80 1280 May 22,2002 May 22, 2002 P7BRQ62 40 640 Jun. 05, 2002 P7BRQ67 Controls (M62)(MCG170) (LeYM12) Mono Ab (S193) (GB81) 320 Mono Ab (VK9) (P7BRQ17)320 >2560 (slovin lab wk 7) Patient Jan. 16, 2002 Jan. 16, 2002 P7BRQ520 20 2 Jan. 23, 2002 Jan. 23, 2002 P7BRQ8 40 0 40 Jan. 30, 2002 Jan.30, 2002 P7BRQ12 2560 160 640 Feb. 13, 2002 P7BRQ20 640 160 1280 Feb.27, 2002 Feb. 27, 2002 P7BRQ27 640 160 1280 Mar. 13, 2002 P7BRQ36 320320 640 Apr. 10, 2002 P7BRQ46 80 160 640 May 22, 2002 May 22, 2002P7BRQ61 160 640 Jun. 05, 2002 P7BRQ68 Controls (P7BRQ17) (hexavalent)(P7BRQ16) (LeYM12) Mono Ab (S193) (GB81) 320 Mono Ab (VK9) (P7BRQ17)320 >2560 (slovin lab wk 7) Patient Jan. 07, 2002 Jan. 07, 2002 P7BRQ1 00 0 3 Jan. 14, 2002 Jan. 14, 2002 P7BRQ3 0 0 0 Jan. 21, 2002 Jan. 21,2002 P7BRQ7 320 0 80 Feb. 04, 2002 P7BRQ17 640 320 2560 Feb. 18, 2002Feb. 18, 2002 P7BRQ23 320 320 1280 Mar. 04, 2002 P7BRQ30 320 1280 640Apr. 01, 2002 P7BRQ41 80 320 320 May 13, 2002 May 13, 2002 P7BRQ57 20160 320 May 27, 2002 P7BRQ63 20 Controls (P7BRQ17) (hexavalent)(P7BRQ16) (LeYM12) Mono Ab (S193) (GB81) 320/640 Mono Ab (VK9) 1280/2560(P7BRQ17) 320 2560 (slovin lab wk 7) Patient Jan. 14, 2002 Jan. 14, 2002P7BRQ2 20 0 40 4 Jan. 21, 2002 Jan. 21, 2002 P7BRQ6 80 0 20 Jan. 28,2002 Jan. 28, 2002 P7BRQ10 320 20 160 Feb. 11, 2002 P7BRQ16  640/1280 40320 Feb. 25, 2002 Feb. 25, 2002 P7BRQ26 320 40 640 Mar. 11, 2002 P7BRQ34320 160 160 did not Apr. 08, 2002 come in Apr. 15, 2002 P7BRQ47 160 160160 May 20, 2002 May 20, 2002 P7BRQ58 40 320 160 Jun. 03, 2002 P7BRQ6640 Controls (P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12) Mono Ab (S193)(GB81) 320/640 Mono Ab (VK9) 1280/2560 (P7BRQ17) 320 >2560 (slovin labwk 7) Patient Feb. 06, 2002 Feb. 06, 2002 P7BRQ15 0 0 0 5 Feb. 13, 2002Feb. 13, 2002 P7BRQ22 0 0 0 Feb. 20, 2002 Feb. 20, 2002 P7BRQ25 0 0 0Mar. 06, 2002 P7BRQ33 20 20 40 Mar. 20, 2002 Mar. 20, 2002 P7BRQ38 0 040 Apr. 03, 2002 P7BRQ42 0 40 40 May 01, 2002 P7BRQ51 20 20 20 Jun. 12,2002 Jun. 12, 2002 P7BRQ71 Jun. 26, 2002 Controls (P7BRQ17) (hexavalent)(P7BRQ16) (LeYM12) Mono Ab (S193) 320/640 (GB81) 1280/2560 Mono Ab (VK9)320 >2560 (P7BRQ17) 320 1280 (slovin lab wk 7) Patient Feb. 06, 2002Feb. 06, 2002 P7BRQ14 0 0 0 6 Feb. 13, 2002 Feb. 13, 2002 P7BRQ18 0 0 0Feb. 20, 2002 Feb. 20, 2002 P7BRQ24 160 160 80 Mar. 06, 2002 P7BRQ31 160160 320 Mar. 20, 2002 Mar. 20, 2002 P7BRQ39 80 40 160 Apr. 03, 2002P7BRQ43 40 160 160 May 01, 2002 P7BRQ52 40 320 320 Jun. 12, 2002 Jun.12, 2002 P7BRQ70 Jun. 26, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16)(LeYM12) Mono Ab (S193) 320/640 (GB81) 1280/2560 Mono Ab (VK9) 320 2550(P7BRQ17) 320 1280 (slovin lab wk 7) ELISA GM2 ELISA MUC-1-1 ELISA LeYCeramide ELISA Globo H (10 mcg) 5G (3 (10 mcg) Ceramide(10 PatientVaccine Sample (April 2002) mcg)(April 2002) (April 2002) mcg) (May2002) Name Date Date Serology Top IgG IgM IgG IgM IgG IgM IgG PatientFeb. 27, 2002 Feb. 27, 2002 P7BRQ29 0 0 0 0 0 0 0 7 Mar. 06, 2002 Mar.06, 2002 P7BRQ32 0 0 0 0 0 0 0 Mar. 13, 2002 Mar. 13, 2002 P7BRQ35 0 2020 320 0 0 0 Mar. 27, 2002 P7BRQ40 0 40 320 320 0 0 0 Apr. 10, 2002 Apr.10, 2002 P7BRQ45 0 80 320 640 0 0 0 Apr. 24, 2002 P7BRQ49 0 80 160 320 00 0 May 22, 2002 P7BRQ60 0 Jul. 03, 2002 Jul. 03, 2002 Controls >2560(P7BRQ17) 320 80 640 (hexavalent) 640 320 0 1280 (P7BRQ16) 0 160(LeYM12) 1280 Mono Ab (S193) 20 (GB81) 0 Mono Ab (VK9) (P7BRQ17) (slovinlab wk 7) (June 2002) (June 2002) (July 2002) (July 2002) Patient Apr.22, 2002 Apr. 22, 2002 P7BRQ48 8 Apr. 29, 2002 Apr. 29, 2002 P7BRQ53 May06, 2002 May 06, 2002 P7BRQ54 May 20, 2002 P7BRQ59 Jun. 03, 2002 Jun.03, 2002 P7BRQ65 Jun. 17, 2002 P7BRQ72 Jul. 15, 2002 Aug. 26, 2002 Aug.26, 2002 Sep. 09, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16)(LeYM12) Mono Ab (S193) (GB81) Mono Ab (VK9) (P7BRQ17) (slovin lab wk 7)Patient Apr. 29, 2002 Apr. 29, 2002 P7BRQ50 9 May 06, 2002 May 06, 2002P7BRQ55 May 13, 2002 May 13, 2002 P7BRQ56 May 27, 2002 P7BRQ64 Aug. 10,2002 Jun. 10, 2002 P7BRQ69 Jun. 24, 2002 Jul. 22, 2002 Sep. 02, 2002Sep. 02, 2002 Sep. 16, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16)(LeYM12) Mono Ab (S193) (GB81) Mono Ab (VK9) (P7BRQ17) (slovin lab wk 7)ELISA Globo H ELISA Tf (10 ELISA dOSM for ELISA Ceramide(10 mcg)(May Tn(3 mcg) sTn Patient Vaccine Sample mcg) (May 2002) 2002) (June 2002) (3mcg) Name Date Date Serology Top IgM IgG IgM IgG IgM IgG IgM PatientFeb. 27, 2002 Feb. 27, 2002 P7BRQ29 0 0 20 7 Mar. 06, 2002 Mar. 06, 2002P7BRQ32 0 0 0 Mar. 13, 2002 Mar. 13, 2002 P7BRQ35 0 320 160 Mar. 27,2002 P7BRQ40 0 640/1280 160 Apr. 10, 2002 Apr. 10, 2002 P7BRQ45 0 640 80Apr. 24, 2002 P7BRQ49 0 1280 80 May 22, 2002 P7BRQ60 0 640 80 Jul. 03,2002 Jul. 03, 2002 Controls (P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12)Mono Ab (S193) 320 (GB81) 1280/2560 Mono Ab (VK9) 640 2560 (P7BRQ17) 6401280 (slovin lab wk 7) Patient Apr. 22, 2002 Apr. 22, 2002 P7BRQ48 8Apr. 29, 2002 Apr. 29, 2002 P7BRQ53 May 06, 2002 May 06, 2002 P7BRQ54May 20, 2002 P7BRQ59 Jun. 03, 2002 Jun. 03, 2002 P7BRQ65 Jun. 17, 2002P7BRQ72 Jul. 15, 2002 Aug. 26, 2002 Aug. 26, 2002 Sep. 09, 2002 Controls(P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12) Mono Ab (S193) (GB81) Mono Ab(VK9) (P7BRQ17) (slovin lab wk 7) Patient Apr. 29, 2002 Apr. 29, 2002P7BRQ50 9 May 06, 2002 May 06, 2002 P7BRQ55 May 13, 2002 May 13, 2002P7BRQ56 May 27, 2002 P7BRQ64 Aug. 10, 2002 Jun. 10, 2002 P7BRQ69 Jun.24, 2002 Jul. 22, 2002 Sep. 02, 2002 Sep. 02, 2002 Sep. 16, 2002Controls (P7BRQ17) (hexavalent) (P7BRQ16) (LeYM12) Mono Ab (S193) (GB81)Mono Ab (VK9) (P7BRQ17) (slovin lab wk 7) Notes Monoclonals: DilutionsVK9 1:200; S193 1:400 DOSM Tested positive by ELISA with Monoclonals 5F4and HB-TN1. OSM Tested positive by ELISA with Monoclonals 5F4, B72.3,and HB-TN1. Shaded region denotes Pre sera (week 1) All samples weretested in duplicate. for patients Lewis and Smith, Lewis Y plates werecoated at 0.3, future plates will be coated at 0.2. Italics controlspreviously tested as positive.

Hexavalent Pilot study, protocol number #00-64, ELISA tested againstTF(c)-HSA Vaccination 1 2 3 4 5 Ag Patient Ab wkl 2 3 7 12 19 21 31 33TFc 1 Igm 40 40 40 40 >1280 40 40 40 40 HSA IgG 20 20 >1280 320 80 80 80160 320 2 IgM NMA 80 160 160 80 40 40 20 20 IgG 10 NMA 10 40 80 NMA NMA20 40 3 Igm 40 IgG NMA 4 IgM 40 20 320 80 80 80 NMA IgG NMA NMA 640 8040 160 320 5 IgM NMA 20 160 20 IgG 10 NMA 10 20 6 IgM 40 640 640 640 16080 80 160 IgG 640  >1280 >1280 >1280 >1280 >1280 >1280 >1280 7 IgM NMA40 80 640 160 80 40 80 80 IgG 10 320  NMA NMA 40 20 80 20 80 8 IgM NMANMA 160 160 IgG NMA NMA 10 80 9 IgM NMA NMA NMA 20 NMA NMA NMA NMA IgGNMA NMA 40 NMA 640 80 >1280 160 10 IgM NMA 10 160 160 IgG NMA NMA 40 2011 IgM 20 IgG NMA 12 Igm 10 40 160 >1280 80 640 80 20 160 IgG NMA NMANMA NMA >640 20 40 40 80 13 IgM NMA IgG NMA 14 IgM 20 80 40 80 80 40 40160 160 IgG NMA 40 160 >1280 640 80 80 80 160 15 IgM NMA NMA 20 80 40 40IgG NMA NMA 10 40 40 40 16 IgM 20 80 >1280 IgG 10 10 80 17 IgM NMA 10NMA 40 40 40 40 40 160 IgG NMA NMA 40 40 80 40 20 40 18 IgM 40 20 80 64040 80 80 20 80 IgG NMA NMA NMA 40 160 40 640 40 40 Positive Controls:IgM(19) 1:1280 IgG (7) 1:640 (−) Human AB Serum As from Oct. 19, 2001end pt. Titers: (+) IgM: 640 IgG: 640 (−) IgM: NMA IgG: NMA

Hexavalent Pilot study, protocol # 00-64 ELISA against glycosylatedMUC1-1 (5 sites) Vaccination 1 2 3 4 5 Ag Patient Number Ab wkl 2 3 7 1219 21 31 MUC 1 Igm NMA 40 >1280 >1280 >1280 >1280 >1280 >1280 1-1G5 IgGNMA NMA 80 160 >1280 640 >1280 >1280 32mer 2 IgM NMA NMA 640 10 NMA 1020 IgG NMA NMA 10 40 40 20.00 40 160 3 Igm 20 640 IgG NMA 40 4 Igm NMANMA 320.00 >1280 >1280 >1280 640 640 IgG NMA NMA 10 320160 >1280 >1280 >1280 5 IgM 10 40 >1280 >1280 IgG NMA NMA 160 >1280 6IgM NMA 320 >1280 320 320 160 80 IgG NMA 160 >1280 320 160 160 160 7 IgMNMA NMA 40 640 640 80 80 40 IgG NMA NMA640 >1280 >1280 >1280 >1280 >1280 8 IgM NMA NMA 160 IgG NMA NMA 10 9 IgMNMA NMA 40 160 80 20 20 10 IgG NMA NMA NMA 40 20 10 80 40 10 Igm NMA NMAIgG NMA NMA 11 IgM NMA NMA 10 40 20 160 160 80 IgG NMA NMA 10 320 160 4040 20 12 Igm 40 40 IgG NMA NMA 13 Igm NMA NMA 20 80 80 320 320 160 IgGNMA NMA 20 20 40 320 320 160 14 Igm 80 20 160 160 40 40 IgG NMA NMA NMANMA 40 20 15 Igm NMA NMA 640 640 640 10 10 10 IgG NMA NMA 640 640 640160 80 80 16 Igm NMA 20 IgG NMA 20 17 IgM NMA 20 320 160 40 640 320 80IgG NMA NMA 10 80 640 40 640 160 Positive Controls: (7) IgM 1:2560 IgG1:2560 Oct. 22, 2001 (−) AB Sera Controls as of Oct. 15, 2001: (+)Igm: >1280 IgG: >1280 (−) IgM: NMA IgG: NMA

Fourth Series of Experiments Polyvalent Conjugate Vaccine for Cancer

Preliminary Data of Vaccination of High Risk Breast Cancer (BC) Patients(Pts) with a Heptavalent Antigen—Keyhole Limpet Hemocyanin (KLH)Conjugate Plus the Immunologic Adjuvant QS-21.

We have previously shown that following vaccination with single antigen(Ag)—KLH conjugates plus QS-21, the majority of BC pts generate specificantibody (AB) titers. (Clin Ca Res 6:1693, 2000; PNAS 98(6):3270, 2001;Proc ASCO 16:439a, 1997, 18:439a, 1999, 20:271a, 2001) Single Ag'stested have included MUC-1 (various peptide lengths), sTn clustered (c),GloboH and GM2. In an effort to improve and broaden the immune response,we treated BC pts with seven Ag's: 10 mcg each of GM2, GloboH, Lewisy;and 3 mcg each of TF(c), sTn(c), Tn(c) and glycosylated MUC-1, (32 aminoacid (aa) sequence, glycosylated at 5 sites per 20 aa tandem repeat).Each Ag was conjugated to KLH and mixed with 100 mcg of QS-21.Heptavalent vaccines were administered subcutaneously during weeks 1, 2,3, 7, and 19. We treated ten patients: median age 48 years (range 43-63yrs); Stage IV=3, Stage 2 with 4 positive nodes=7. Nine pts havecompleted immunization. Toxicity was limited to transient grade 2 localskin reactions and grade 1-2 flu-like symptoms. IgM and IgG AB titerswere considered positive for each antigen if there was at least aneightfold increase above baseline more than once, during weeks 1-19.Antibody responses are tabulated. (table) MUC1 and TF(c) seem mostimmunogenic. Flow cytometric analysis (FACS) was obtained pre and posttherapy to detect binding of IgM and IgG AB against MCF-7 tumor cells. Apositive FACS was defined as at least a threefold increase abovebaseline and was observed in 6/9 patients for IgM and 0/9 for IgG.Further analyses are ongoing. Our next cohort will evaluate the sameantigens conjugated to KLH but with GP-100 as the immunologic adjuvant.

Number of pts with positive AB response/Number of pts evaluable

Ag GM2 Ley MUC1 TF(c) sTn(c) Tn(c) GloboH IgM 2/9 1/9 8/9 8/9 4/9 7/96/9 IgG 0/9 1/9 8/9 8/9 1/9 0/9 0/9

Objectives

-   -   Determine immune response against seven antigens and cell lines        expressing these antigens    -   Evaluate toxicity

Background

-   -   Preclinical data demonstrates that conjugation of an antigen        with keyhole limpet hemocyanin (KLH) and addition of the immune        adjuvant QS-21 augments immunogenicity (Cancer Immunol        Immunother 41:185, 1985; Cancer Res 56:3315, 1996)    -   Following vaccination with single antigen—KLH conjugates plus        QS-21, most breast cancer patients generated IgM and IgG        antibodies against the immunizing antigens (Clin Ca Res 6:1693,        2000; PNAS 98(6):3270, 2001; Proc ASCO 16:439a, 1997, 18:439a.        1999, 20:271a, 2001)    -   These single antigens have included MUC-1 (various peptide        lengths), sTn clustered (c), GloboH and GM2    -   To broaden the immune response, seven antigens were individually        conjugated to KLH and mixed with QS-21 to construct this        heptavalent vaccine

Vaccine Components

-   -   Antigens        -   Protein: glycosylated MUC-1 (32aa peptide)        -   Gangliosides: GM2, GloboH        -   Carbohydrates: LewisY, sTn(c), Tn(c),TF(c)    -   Immunogenic protein carrier        -   KLH (by the following methods of conjugation):            -   MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)                linker for TF(c), sTn(c), Tn(c), and MUC-1            -   MMCCH (4-[4-N-maleimidomethyl] cyclohexane-1-carboxyl                hydrazide) linker for GloboH and Le^(Y)            -   Direct reductive amination for GM2    -   Immunologic Adjuvant        -   QS-21 (purified saponin fraction of tree bark)

Vaccine Components

Doses Antigens* GM2 10 mcg MUC-1 3 mcg LewisY 10 mcg GloboH 10 mcg TF(c)3 mcg Tn(c) 3 mcg sTn (c) 3 mcg Adjuvant QS-21 100 mcg *(each conjugatedto KLH)

Treatment and Evaluation Plan

WEEK # 1 2 3 5 7 9 13 19 21 (q 3 months) VACCINE 1 2 3 4 5 Blood ✓ ✓ ✓ ✓✓ ✓ ✓ ✓ ✓ ✓ Samples for Immune Response

Eligibility Criteria

Breast cancer patients with any one of the following features:

-   -   Stage 1V (stable on hormone therapy [tx])    -   Stage 1V (no evidence of disease [NED])    -   Stage III    -   Stage II (≧4 positive nodes)    -   Ipsilateral breast or axillary recurrence    -   Rising CA15-3 or CEA levels and NED

Patient Characteristics

-   -   Total number of patients treated: 10    -   Total number of vaccinations completed: 50    -   *(One patient was delayed for unrelated issues)    -   Median age: 48 years (range 43-63 years)    -   Stage

II (with ≧4+ nodes): 7 IV (NED): 2 IV (stable on hormone tx): 1 n = 10

Common Toxicities

-   -   Grade 1-2 injection site skin reactions    -   Grade 1-2-flu-like symptoms    -   No significant laboratory abnormalities    -   No definite autoimmune reactions

Response Criteria: ELISA

-   -   Serologic Response by ELISA (Enzyme-Linked Immunosorbent Assay)        -   IgM and IgG antibody titers were considered positive for            each antigen if there was a eightfold increase above            baseline more than once, during weeks 1-19    -   Immunologic Response    -   Patient was considered a responder if there was a serologic        response to at least 3 of the 7 antigens Response Criteria: FACS        and CDC    -   Response by FACS (fluorescence activated cell sorter) was        considered positive if there was the following increase above        baseline:        -   ≧3-fold increase in percent gated positivity, AND        -   ≧1.5-fold increase on MFI (mean fluorescence intensity)    -   Response by CDC (complement-dependent cytotoxicity) was        considered positive if there was a 20% increase above baseline

Immune Response Data ELISA

GM2 MUC-1 Lewisy GLOBOH TF Tn sTn IgM IgG IgM IgG IgM IgG IgM IgG IgMIgG IgM IgG IgM IgG 1 − − + + − − − − + + − − − − 2 + − + + − − +− + + + − + − 3 + − + + − − + − + + + − + − 4 − − + + + − + + + + + − −− 5 − − − + − − − − − − − − − − 6 − − + + − − + − + + + − + − 7 + − + +− − − − + + + − − − 8 − − + + − − + − + + − − + − 9 − − + + − − + − + +− − − + 10  − − + + − − + − + + − − + − ·SUM 3 0 9 10 1 0 7 1 9 9 5 0 51

Immune Response Data FACS and CDC

FACS (IgM) CDC Patient MCF-7 LSC Du-175 MCF-7 LSC 1 + + + − − 2 − +− + + 3 + − − − − 4 + + + + + 5 − − − − − 6 + + − + − 7 + + − − + 8 − +− + − 9 + + + − − 10  − − − + − • SUM 6 7 3 5 3

CONCLUSION

-   -   Vaccination with a heptavalent antigen-KLH conjugate plus QS-21        is well tolerated in breast cancer patients    -   IgM and IgG antibody responses (to at least 3 of 7 antigens)        were observed in 8 patients and 2 patients respectively    -   MUC1 and TF(c) to be appear the most immunogenic of the seven        antigens in this vaccine    -   IgM antibody binding to tumor cells (MCF-7, LSC, Du-145) by FACS        analysis was observed in 6 patients, 7 patients, and 3 patients        respectively    -   There was no consistent evidence of IgG antibody binding to        tumor cells by FACS    -   There was evidence of CDC with the MCF-7 and LSC tumor cell        lines in 5 patients and 3 patients respectively    -   Our next cohort will evaluate the same antigens conjugated to        KLH but with GP-100 as the immunologic adjuvant

1-50. (canceled)
 51. An immunogenic composition comprising an adjuvantand antigens comprising MUC-1, TN, GloboH, TF(c) and at least oneantigen selected from the group consisting of sTn(c), Tn(c) and sialylLe^(a), wherein said antigens are individually conjugated to a carrier.52. The immunogenic composition of claim 51, wherein said antigenscomprise MUC-1, TN, GloboH, TF(c) and sTn(c).
 53. The immunogeniccomposition of claim 51, wherein said antigens comprise MUC-1, TN,GloboH, TF(c) and Tn(c).
 54. The immunogenic composition of claim 51,wherein said antigens comprise MUC-1, TN, GloboH, TF(c) and sialylLe^(a).
 55. The immunogenic composition of claim 51, wherein at leasttwo antigens are selected from the group consisting of sTn(c), Tn(c) andsialyl Le^(a).
 56. The immunogenic composition of claim 55, wherein saidat least two antigens are sTn(c) and Tn(c).
 57. The immunogeniccomposition of claim 55, wherein said at least two antigens are sTn(c)and sialyl Le^(a).
 58. The immunogenic composition of claim 55, whereinsaid at least two antigens are Tn(c) and sialyl Le^(a).
 59. Theimmunogenic composition of claim 51, wherein said antigens compriseMUC1, Tn, GloboH, TF(c), sTn(c), Tn(c) and sialyl Le^(a).
 60. A methodfor inducing antibody production in a subject comprising administeringan immunogenic composition of claim
 51. 61. An immunogenic compositioncomprising an adjuvant and antigens comprising MUC1-Tn conjugate,GloboH, TF(c) and at least one antigen selected from the groupconsisting of sTn(c), Tn(c) and sialyl Le^(a), wherein said conjugateand each of said antigens are individually conjugated to a carrier. 62.The immunogenic composition of claim 61 wherein said antigens compriseMUC1-Tn conjugate, GloboH, TF(c) and sTn(c).
 63. The immunogeniccomposition of claim 61 wherein said antigens comprise MUC1-Tnconjugate, GloboH, TF(c) and Tn(c).
 64. The immunogenic composition ofclaim 61 wherein said antigens comprise MUC1-Tn conjugate, GloboH, TF(c)and sialyl Le^(a).
 65. The immunogenic composition of claim 61, whereinat least two antigens are selected from the group consisting of sTn(c),Tn(c) and sialyl Le^(a).
 66. The immunogenic composition of claim 65,wherein said at least two antigens are sTn(c) and Tn(c).
 67. Theimmunogenic composition of claim 65, wherein said at least two antigensare sTn(c) and sialyl Le^(a).
 68. The immunogenic composition of claim65, wherein said at least two antigens are Tn(c) and sialyl Le^(a). 69.The immunogenic composition of claim 61, wherein said antigens compriseMUC1-Tn conjugate, GloboH, TF(c), sTn(c), Tn(c) and sialyl Le^(a).
 70. Amethod for inducing antibody production in a subject comprisingadministering an immunogenic composition of claim
 61. 71. A method forinducing antibody production in a subject comprising administering animmunogenic composition comprising antigens comprising MUC1-Tnconjugate, GloboH and TF(c), wherein said conjugate and each of saidantigens are individually conjugated to a carrier.